Xenopus CAF1 requires NOT1-mediated interaction with 4E-T to repress translation in vivo

Abstract

RNA-regulatory factors bound to 3 ′ UTRs control translation and stability. Repression often is associated with poly(A) removal. The deadenylase CAF1 is a core component of the CCR4-NOT complex. Our prior studies established that CAF1 represses translation independent of deadenylation. We sought the mechanism of its deadenylation-independent repression in Xenopus oocytes. Our data reveal a chain of interacting proteins that links CAF1 to CCR4-NOT and to Xp54 and 4E-T. Association of CAF1 with NOT1, the major subunit of CCR4-NOT, is required for repression by CAF1 tethered to a reporter mRNA. Affinity purification-mass spectrometry and coimmunoprecipitation revealed that at least five members of the CCR4-NOT complex were recruited by CAF1. The recruitment of these proteins required NOT1, as did the ability of tethered CAF1 to repress translation. In turn, NOT1 was needed to recruit Xp54 and 4E-T. We examined the role of 4E-T in repression using mutations that disrupted either eIF4E-dependent or -independent mechanisms. Expression of a 4E-T truncation that still bound eIF4E alleviated repression by tethered CAF1, NOT1, and Xp54. In contrast, a mutant 4E-T that failed to bind eIF4E did not. Repression of global translation was affected only by the eIF4E-dependent mechanism. Reporters bearing IRES elements revealed that repression via tethered CAF1 and Xp54 is cap-and eIF4E-independent, but requires one or more of eIF4A, eIF4B, and eIF4G. We propose that RNA-binding proteins, and perhaps miRNAs, repress translation through an analogous chain of interactions that begin with the 3 ′ UTR-bound repressor and end with the noncanonical activity of 4E-T.

Cap-binding protein 4EHP effects translation silencing by microRNAs

“…2D). Importantly, the middle domain also interacts with 4E-T via DDX6 (9,10,(14)(15)(16). To investigate whether 4EHP plays a role in translation repression by the CCR4-NOT complex, we used the λN-BoxB tethering approach (35).…”

“…The silencing activity of miRISC is mediated by the CCR4-NOT deadenylase complex through the scaffolding subunit, CNOT1 (6)(7)(8). CNOT1 recruits the DDX6 and 4E-T (eIF4E transporter, also known as EIF4ENIF1) proteins, which are important for miRNA-mediated silencing (9)(10)(11)(12)(13)(14)(15)(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 Functions of MicroRNAs: mRNA Decay and Translational Repression

“…Although it is still unclear exactly how miRNAs repress translation, three major mechanisms have been proposed in the past few years, including (i) GW182-mediated PABP displacement [48,49], (ii) recruitment of the translational repressors through GW182 [45,46,55,61,83,84], and (iii) dissociation of eIF4A from the cap-binding complex eIF4F [85,86]. These mechanisms are not mutually exclusive; they may overlap, occur concurrently, or occur with different kinetics to augment the overall silencing effect ( Figure 3).…”

“…In mouse embryonic stem cells, DDX6 was shown to be recruited onto miRNA targets through its interaction with the mammalian hyperplastic discs protein EDD, which binds to GW182 through the PABC domain [44]. Recent studies suggest that the eIF4E-binding protein 4E-T is recruited onto the CCR4-NOT complex through DDX6, PATL1, or LSM14 [55,84]. 4E-T represses general translation through the eIF4E-binding domain [84,102].…”

“…Recent studies suggest that the eIF4E-binding protein 4E-T is recruited onto the CCR4-NOT complex through DDX6, PATL1, or LSM14 [55,84]. 4E-T represses general translation through the eIF4E-binding domain [84,102]. However, tethering of 4E-T repressed translation of the target mRNA in a manner independent of its 4E-binding domain in human cell lines and Xenopus oocyte [84,102].…”