An eIF4E1/4E-T complex determines the genesis of neurons from precursors by translationally repressing a proneurogenic transcription program
ABSTRACT: Here, we have addressed the mechanisms that determine genesis of the correct numbers of neurons during development, focusing upon the embryonic cortex. We identify in neural precursors a repressive complex involving eIF4E1 and its binding partner 4E-T that coordinately represses translation of proteins that determine neurogenesis. This eIF4E1/4E-T complex is present in granules with the processing body proteins Lsm1 and Rck, and disruption of this complex causes premature and enhanced neurogenesis and neural precursor depletion. Analysis of the 4E-T complex shows that it is highly enriched in mRNAs encoding transcription factors and differentiation-related proteins. These include the proneurogenic bHLH mRNAs, which colocalize with 4E-T in granules, and whose protein products are aberrantly upregulated following knockdown of eIF4E, 4E-T, or processing body proteins. Thus, neural precursors are transcriptionally primed to generate neurons, but an eIF4E/4E-T complex sequesters and represses translation of proneurogenic proteins to determine appropriate neurogenesis. We obtained 3 biological replicates of IgG-bound RNA, 4E-T-bound RNA, and cooresponding total RNA input from mouse E12-13 cortices. RNA samples were analyzed on the Affymetrix Mouse Gene 2.0 ST Arrays.
Project description:Here, we have addressed the mechanisms that determine genesis of the correct numbers of neurons during development, focusing upon the embryonic cortex. We identify in neural precursors a repressive complex involving eIF4E1 and its binding partner 4E-T that coordinately represses translation of proteins that determine neurogenesis. This eIF4E1/4E-T complex is present in granules with the processing body proteins Lsm1 and Rck, and disruption of this complex causes premature and enhanced neurogenesis and neural precursor depletion. Analysis of the 4E-T complex shows that it is highly enriched in mRNAs encoding transcription factors and differentiation-related proteins. These include the proneurogenic bHLH mRNAs, which colocalize with 4E-T in granules, and whose protein products are aberrantly upregulated following knockdown of eIF4E, 4E-T, or processing body proteins. Thus, neural precursors are transcriptionally primed to generate neurons, but an eIF4E/4E-T complex sequesters and represses translation of proneurogenic proteins to determine appropriate neurogenesis. We obtained 3 biological replicates of IgG-bound RNA, 4E-T-bound RNA, and cooresponding total RNA input from mouse E12-13 cortices. RNA samples were analyzed on the Affymetrix Mouse Gene 2.0 ST Arrays.
Project description:UNLABELLED:Here, we have asked about post-transcriptional mechanisms regulating murine developmental neurogenesis, focusing upon the RNA-binding proteins Smaug2 and Nanos1. We identify, in embryonic neural precursors of the murine cortex, a Smaug2 protein/nanos1 mRNA complex that is present in cytoplasmic granules with the translational repression proteins Dcp1 and 4E-T. We show that Smaug2 inhibits and Nanos1 promotes neurogenesis, with Smaug2 knockdown enhancing neurogenesis and depleting precursors, and Nanos1 knockdown inhibiting neurogenesis and maintaining precursors. Moreover, we show that Smaug2 likely regulates neurogenesis by silencing nanos1 mRNA. Specifically, Smaug2 knockdown inappropriately increases Nanos1 protein, and the Smaug2 knockdown-mediated neurogenesis is rescued by preventing this increase. Thus, Smaug2 and Nanos1 function as a bimodal translational repression switch to control neurogenesis, with Smaug2 acting in transcriptionally primed precursors to silence mRNAs important for neurogenesis, including nanos1 mRNA, and Nanos1 acting during the transition to neurons to repress the precursor state. SIGNIFICANCE STATEMENT:The mechanisms instructing neural stem cells to generate the appropriate progeny are still poorly understood. Here, we show that the RNA-binding proteins Smaug2 and Nanos1 are critical regulators of this balance and provide evidence supporting the idea that neural precursors are transcriptionally primed to generate neurons but translational regulation maintains these precursors in a stem cell state until the appropriate developmental time.
Project description:4E-Transporter binds eIF4E via its consensus sequence YXXXXL?, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.
Project description:Stress granules (SGs) arise as a consequence of cellular stress, contain stalled translation preinitiation complexes, and are associated with cell survival during environmental insults. SGs are dynamic entities with proteins relocating into and out of them during stress. Among the repertoire of proteins present in SGs is eukaryotic initiation factor 4E (eIF4E), a translation factor required for cap-dependent translation and that regulates a rate-limiting step for protein synthesis. Herein, we demonstrate that localization of eIF4E to SGs is dependent on the presence of a family of repressor proteins, eIF4E-binding proteins (4E-BPs). Our results demonstrate that 4E-BPs regulate the SG localization of eIF4E.
Project description:Human 4E-T is an eIF4E-binding protein (4E-BP) present in processing (P)-bodies that represses translation and regulates decay of mRNAs destabilized by AU-rich elements and microRNAs (miRNAs). However, the underlying regulatory mechanisms are still unclear. Here, we show that upon mRNA binding 4E-T represses translation and promotes deadenylation via the recruitment of the CCR4-NOT deadenylase complex. The interaction with CCR4-NOT is mediated by previously uncharacterized sites in the middle region of 4E-T. Importantly, mRNA decapping and decay are inhibited by 4E-T and the deadenylated target is stored in a repressed form. Inhibition of mRNA decapping requires the interaction of 4E-T with the cap-binding proteins eIF4E/4EHP. We further show that regulation of decapping by 4E-T participates in mRNA repression by the miRNA effector protein TNRC6B and that 4E-T overexpression interferes with tristetraprolin (TTP)- and NOT1-mediated mRNA decay. Thus, we postulate that 4E-T modulates 5'-to-3' decay by swapping the fate of a deadenylated mRNA from complete degradation to storage. Our results provide insight into the mechanism of mRNA storage that controls localized translation and mRNA stability in P-bodies.
Project description:4E-BP (eIF4E-BP) represses translation initiation by binding to the 5' cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity, and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used HyperTRIBE (targets of RNA binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets more substantially compared to nontargets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.
Project description: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.
Project description:The mechanisms instructing genesis of neuronal subtypes from mammalian neural precursors are not well-understood. To address this issue, we have characterized the transcriptional landscape of radial glial precursors (RPs) in the embryonic murine cortex. We show that individual RPs express mRNA but not protein for transcriptional specifiers of both deep and superficial layer cortical neurons. Some of these mRNAs, including the superficial versus deep layer neuron transcriptional regulators Brn1 and Tle4, are translationally repressed by their association with the RNA-binding protein Pumilio2 and the 4E-T protein. When these repressive complexes are disrupted in RPs mid-neurogenesis by knocking down 4E-T or Pum2, this causes aberrant co-expression of deep layer neuron specification proteins in newborn superficial neurons. Thus, cortical RPs are transcriptionally primed to generate diverse types of neurons, and a 4E-T-Pum2 complex represses translation of some of these neuronal identity mRNAs to ensure appropriate temporal specification of daughter neurons. Overall design: We obtained 3 biological replicates of IgG-bound RNA, Pum2-bound RNA, and corresponding total RNA input from E12/E13 mouse cortices. RNA samples were analyzed on Mouse Gene 2.0 ST Arrays (Thermo Fisher)
Project description:A key player in translation initiation is eIF4E, the mRNA 5' cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4L site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4L, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.
Project description:In addition to the canonical eIF4E cap-binding protein, eukaryotes have evolved sequence-related variants with distinct features, some of which have been shown to negatively regulate translation of particular mRNAs, but which remain poorly characterised. Mammalian eIF4E proteins have been divided into three classes, with class I representing the canonical cap-binding protein eIF4E1. eIF4E1 binds eIF4G to initiate translation, and other eIF4E-binding proteins such as 4E-BPs and 4E-T prevent this interaction by binding eIF4E1 with the same consensus sequence YX 4L?. We investigate here the interaction of human eIF4E2 (4EHP), a class II eIF4E protein, which binds the cap weakly, with eIF4E-transporter protein, 4E-T. We first show that ratios of eIF4E1:4E-T range from 50:1 to 15:1 in HeLa and HEK293 cells respectively, while those of eIF4E2:4E-T vary from 6:1 to 3:1. We next provide evidence that eIF4E2 binds 4E-T in the yeast two hybrid assay, as well as in pull-down assays and by recruitment to P-bodies in mammalian cells. We also show that while both eIF4E1 and eIF4E2 bind 4E-T via the canonical YX 4L? sequence, nearby downstream sequences also influence eIF4E:4E-T interactions. Indirect immunofluorescence was used to demonstrate that eIF4E2, normally homogeneously localised in the cytoplasm, does not redistribute to stress granules in arsenite-treated cells, nor to P-bodies in Actinomycin D-treated cells, in contrast to eIF4E1. Moreover, eIF4E2 shuttles through nuclei in a Crm1-dependent manner, but in an 4E-T-independent manner, also unlike eIF4E1. Altogether we conclude that while both cap-binding proteins interact with 4E-T, and can be recruited by 4E-T to P-bodies, eIF4E2 functions are likely to be distinct from those of eIF4E1, both in the cytoplasm and nucleus, further extending our understanding of mammalian class I and II cap-binding proteins.