Fig 1: eIF4A2 acts as both a translation activator and repressor of specific mRNAs encoding cellular potency factors.(A) Flow cytometry for OP-puro (O-propargyl-puromycin) incorporation in ESCs with control KD and eIF4A2 KD. CHX, cycloheximide, a protein synthesis inhibitor. (B) Schematic diagram of the eCLIP-seq protocol. WB, western blot. (C) Peak distribution around mRNAs of eIF4A2 targets, identified by eCLIP-seq. (D) GO analysis of eIF4A2 targets identified by eCLIP-seq. (E) Schematic diagram of SILAC-MS experiment protocol. (F) Graph of the frequency distribution of heavy/light (H/L; eIF4A2 KD/control) ratios of all proteins identified by SILAC-MS, with GO analysis of the genes that are targeted by eIF4A2 with decreased (left side, blue box) or increased (right side, orange box) protein levels while maintaining mRNA levels upon eIF4A2 KD. (G) Schematic diagram of the ribosome profiling protocol. (H) IGV (Integrative Genomics Viewer) snapshots on candidate genes showing RNA-seq (red) and RPF profiling (purple) datasets between control (shCtrl) and eIF4A2 (sh4A2) KD, and eIF4A2 binding (eCLIP-seq) profiling data (green) with binding of eIF4A2 at TIRs (for Nanog, Oct4, and Sox2) and the CDS of Zscan4c near 3′UTR shaded in green. The green marks below the Zscan4c gene architecture diagram are the CLIP–quantitative polymerase chain reaction (qPCR) amplicon positions in Fig. 6F. (I) Western blots of Nanog, Oct4, Sox2, and Zscan4 in ESCs with control KD and eIF4A2 KD. Vinculin serves as the loading control. (J) A model depicting eIF4A2-mediated translation activation of pluripotency transcripts and repression of Zscan4 in ESCs.
Fig 2: eIF4A2 represses the 2C gene Zscan4 through the interaction with Ddx6.(A) Depiction of the double-reporter system with Zscan4c-EGFP and MERVL-tdTomato. (B and C) The flow cytometry (B) and the quantification (C) results of indicated populations in single and double KD cells as shown. (D) Depiction of the eIF4A2 interactome with the top two GO terms and associated proteins. Poly(A), polyadenylate. (E, H, L, M, and N) Western blots of the indicated proteins under treatments as indicated. Vinculin serves as the loading control. The protein quantification in (N) is normalized to the Vinculin protein density in (M). (F) CLIP–quantitative PCR on Zscan4c with eIF4A2, Ddx6, or immunoglobulin G (IgG) pulldown. The amplicon positions are labeled in Fig. 2H. (G) IGV snapshots on Zscan4c/d showing RNA-seq data (top two), polysome profiling data for monosome (mono), low polysome (low), and high polysome (high) (middle three and bottom three) between control WT (middle three) and Ddx6 KO (bottom three). RNA-seq and polysome profiling data are from GSE112765 and GSE112761 (38). (I) GSEA result of the 2C-like ESCs (Z4 event, Zscan4 expression) gene set by comparing Ddx6 KO with control cells. (J) qRT-PCR of Zscan4c in shNT-, shDdx6-, or sheIF4A2-infected ESCs with the treatment of the transcription inhibitor DRB (5,6-dichlorobenzimidazole 1-β-d-ribofuranoside, 100 μM) at different time points. (K) IGV snapshots on Ddx6 with similar datasets are shown in Fig. 2H. (O) A model depicting eIF4A2-mediated translation initiation activation of Ddx6 and the cooperation between eIF4A2 and Ddx6 in the inhibition of Zscan4 in ESCs.
Fig 3: eIF4A2 localises to arsenite-induced stress granules but not to hippuristanol-induced stress granules. HeLa cells were cultured and either left untreated or subjected to the following treatments, 1 mM sodium arsenite (AR) for 30 min, heat-shock (HS) at 42°C for 30 min, 1 μM hippuristanol (HP) for 60 min or 3 Gy ionising radiation (IR) recovery for 30 min. The cells were fixed and immunostained for both eIF4A2 (as in Fig. 3) and TIA-1, followed by DAPI. Merged images show TIA-1 (red), eIF4A2 (green) and DAPI (blue). Scale bars: 10 μm.
Fig 4: An RNAi screen identifies the requirement of eIF4A2 for maintaining the ESC identity.(A) Schematic of the eukaryotic cap–dependent translation initiation process. PABP, polyadenylate-binding protein; Ncbp1, nuclear cap binding protein subunit 1. (B) Schematic of the RNAi screen to identify TIF dependency in ESCs. Puro+ indicates that the shRNA plasmid contains a puromycin-resistant gene. AP is alkaline phosphatase. (C) The RNAi screen results. The box colors denote ESC states as indicated. The selected candidates are highlighted in yellow. sh1 to sh3 are three short hairpins for each gene, and screening was performed in biological replicates. (D) Representative examples of the AP-stained colony results from the RNAi screen, including the positive controls (shNanog and shSox2) and the negative control (shGFP). The border colors match the box colors used in (C). (E) Proliferation curves for ESCs with control KD (shNT and shGFP), eIF4A2 KD (sheIF4A2), eIF4A2 KD rescued with WT eIF4A2 (sheIF4A2 + WT), or helicase activity mutants of eIF4A2 (sheIF4A2 + DQAD/NEAD). (F and G) Heatmap (F) and Gene Ontology (GO) analysis (G) of the up-regulated and down-regulated transcripts upon eIF4A2 KD from RNA-seq data. shLuc, shLuciferase; EV, empty vector control. (H) GSEA results of primary germ layer gene sets (epidermis development, mesoderm development, endoderm differentiation, and formation of primary germ layer), placenta (trophectoderm) gene set, and 2C-like ESCs gene set (Z4 event, Zscan4 expression) by comparing eIF4A2 KD with control KD cells from RNA-seq data. NES, normalized enrichment score. (I) Examples of the RNA-seq result in multiple groups. shCtrl (control shRNA) includes shLuc and shEV control KD experiments.
Fig 5: A model depicting eIF4A2-mediated translational control in safeguarding ESC identity.eIF4A2 is responsible for a unique translation initiation control network dedicated to safeguarding ESC identity. (A) eIF4A2 binds to the TIR of its targets to activate the translation initiation: eIF4A2 activates the translation initiation of H3.3 and Rps26 through Rps26-dependent ribosomes (red); eIF4A2 also activates specific pluripotency-associated mRNAs and Ddx6 through Rps26-independent ribosomes (blue). (B) Via the physical interaction with Ddx6, eIF4A2 represses Zscan4’s expression by binding CDS near 3′UTR (non-TIR) of Zscan4 mRNAs (orange).
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