Fig 2: eIF1A interacts with Ago2(a) Proteins co-immunoprecipitated with Flag-HA-Ago2 from HEK293 cells were separated by SDS-PAGE (silver staining) and analyzed by mass spectrometry. eIF1A is a novel Ago2 interacting protein (*:detected peptides of eIF1A). (b) Fluorescence immunostaining was performed with anti-eIF1A (monoclonal) and anti-Ago2 (monoclonal) antibodies and DAPI (4′,6-diamidino-2-phenylindole) in HEK293 and human glioblastoma multiforme T98G cells. White arrows show the co-localized eIF1A and Ago2. (c) eIF1A interacts with Ago2. Human eIF1A was transiently overexpressed in stable HEK293 cells stably expressing Agos (1~4). Immunoprecipitation assays were performed with anti-eIF1A (monoclonal) antibody and immuno blotting (IB) with anti-Agos antibodies in the upper panel and anti-eIF1A antibodies in the bottom panel.

Fig 3: Western blot analysis of MRPL18 (A), TIMM8B (B), and EIF1A (C) from in vitro experiments using a normal colon mucosa epithelial cell line exposed to high (HG) or NG condition. Relative densitometric quantification and representative immunoblots are shown. CO: (osmotic) control. Error bars indicate SEM. Mann–Whitney statistical U‐test used with three biologically independent replicates included (*P‐value < 0.05).

Fig 4: eIF1A binds to MID-domain of Ago2 in a RNA-binding-independent manner(a) Human eIF1A interacts with the MID fragment of human Ago2. Recombinantly expressed and Benzonase-treated Ago2 fragments and recombinantly expressed GST-eIF1A were used for GST-pull down analyses followed by Western blotting with anti-His antibodies (IB, immuno-blotting). The arrow shows that the MID fragment interacts with eIF1A. (b) The globular domain (GD) of eIF1A interacts with Ago2. Recombinantly expressed and Benzonase-treated GST, GST-eIF1A and mutants (ND, 25 ~ 144aa; CD, 1 ~ 114aa; GD, 25 ~ 114aa) were used for GST-pull down assays with cell lysates of HEK293 stably expressing Ago2. (c) 1H-15N TROSY-HSQC spectrum of 15N-eIF1A titrated with the sumo-MID (432 ~ 575aa). The titration of RNA-free-sumo-MID leads to broadening of RNA free 15N-eIF1A resonances (molar ratio of 1:1). Inserted frames show the broadening of resonances of V55, K56 and K67 residues in eIF1A upon MID titration. (d) 1H-15N TROSY-HSQC spectrum of 15N-Sumo-MID titrated with eIF1A. The titration of eIF1A (molar ratio of 1:1) results in broadening 1H-15N TROSY-HSQC crosspeaks coming from the 15N-MID domain, but not from 15N-Sumo (SMT3) tag. Inserted frames show the broadening of resonances of residues from MID-domain, but not from SMT3-Tag (SUMO-tag), upon eIF1A titration.

Fig 5: Schematic diagram for eIF1A functions in Ago2-dependent miR-451 biogenesis, RNA interference and erythrocyte maturation in zebrafishThe primary miRNA (priRNA) is cleaved by Drosha-DGCR8 pathways to generate pre-miRNA within the nucleus. Exportin-5 in complex with Ran-GTP exports the pre-miRNA to the cytoplasm, where the pre-miRNA is bound by DICER to form a RISC Loading Complex that includes Ago2. In the canonical miRNA biogenesis pathway, DICER removes the terminal loop region to yield the mature miRNA. The pre-miR-451 is loaded directly onto Ago2 and sliced on the 3′ hairpin arm. The miR-144 biogenesis is DICER-dependent. The globular domain (GD) of eIF1A directly binds to MID-domain of Ago2 and forms an eIF1A-Ago2 complex promoting Ago2-mediated RNAi and miR-451 biogenesis. MID-domain of Ago2 binds to the GD of eIF1A and does not impair eIF1A functions in translation initiation. The 5′-term of guide strand of miRNA-miRNA* duplex is docked onto a pocket with residues mainly from MID-domain. The long and structured mRNAs are scanned by RISCs to recognize seed regions in miRNAs. After perfect or imperfect complementary guide with miRNAs, mRNAs are nicked by PIWI domain resulting in RNAi. eIF1A augments Ago2-mediated RNAi, miR-451 production and erythrocyte maturation. Black arrows are from previous reports of other groups14,15,43; blue arrows are from this study.