Cap-binding protein 4EHP effects translation silencing by microRNAs

Abstract

MicroRNAs (miRNAs) play critical roles in a broad variety of biological processes by inhibiting translation initiation and by destabilizing target mRNAs. The CCR4-NOT complex effects miRNA-mediated silencing, at least in part through interactions with 4E-T (eIF4E transporter) protein, but the precise mechanism is unknown. Here we show that the cap-binding eIF4E-homologous protein 4EHP is an integral component of the miRNA-mediated silencing machinery. We demonstrate that the cap-binding activity of 4EHP contributes to the translational silencing by miRNAs through the CCR4-NOT complex. Our results show that 4EHP competes with eIF4E for binding to 4E-T, and this interaction increases the affinity of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP interaction engenders a closed-loop mRNA conformation that blocks translational initiation of miRNA targets.M icroRNAs (miRNAs) are short, ∼22-nucleotide noncoding RNAs that affect gene expression in most eukaryotes. miRNAs mediate posttranscriptional silencing by guiding the miRNA-induced silencing complex (miRISC), an assembly of Argonautes and GW182/TNRC6 proteins, to target mRNAs. Target recognition initiates a succession of events: mRNA translational repression, deadenylation, and mRNA decay (1). miRNAs impair the function of eIF4F, a three-subunit complex composed of eIF4E, the m 7 GTP (cap)-interacting factor; eIF4G, a scaffolding protein; and eIF4A, a DEAD-box RNA helicase (2-5). The silencing activity of miRISC is mediated by the CCR4-NOT deadenylase complex through the scaffolding subunit, CNOT1 (6-8). CNOT1 recruits the DDX6 and 4E-T (eIF4E transporter, also known as EIF4ENIF1) proteins, which are important for miRNA-mediated silencing (9-16). The 4E-T protein is a conserved eIF4E-binding protein, which directly binds to the dorsal surface of eIF4E through its canonical eIF4E-binding YX 4 LL (Y 30 TKEELL) motif and impairs the eIF4E/eIF4G interaction and translation initiation (17). The 4E-T protein also facilitates the decay of CCR4-NOT-targeted mRNAs by linking the 3′-terminal mRNA decay machinery to the cap via its interaction with eIF4E (13).In mammals, eIF4E is the best-studied member of a family of proteins composed of eIF4E (eIF4E1), 4EHP (4E-homologous protein; eIF4E2), and eIF4E3. The 4EHP and eIF4E proteins share 28% sequence identity (18,19). The 4EHP protein is ubiquitously expressed, and it is 5-10 times less abundant than eIF4E in a number of mammalian cell lines (18)(19)(20). Like eIF4E, 4EHP binds to 4E-T, but in sharp contrast to eIF4E, it does not associate with eIF4G (18, 21). The 4EHP protein has a 30-to 100-fold weaker affinity for the cap than eIF4E due to a twoamino acid substitution in its cap-binding pocket (22).The 4EHP protein has primarily been documented as a translation repressor. In the Drosophila embryo, 4EHP associates with the RNA binding protein Bicoid to repress caudal mRNA translation (23). Similarly, 4EHP also represses the hunchback mRNA by binding to the nanos repressive element complex, which consists of nanos...

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The suppressive cap-binding-complex factor 4EIP is required for normal differentiation

“…Mammalian and Drosophila 4EHPs are required for correct translation control during development (18)(19)(20)(21). Interactions with partner proteins such as Bicoid (16) or GIGYF2 (=GIF2) (22)(23)(24), and with the RISC complex (25,26), have been implicated in induction of mRNA decay and/or translation suppression by 4EHPs. GIGYF2 appears to be able to act both in conjunction with (24), and independently of, 4EHP (27), and interactions with both Ago2 (28) and the CAF1/NOT complex (27) have been documented.…”

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