A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay - PubMed (original) (raw)

A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay

Maria A Ferraiuolo et al. Proc Natl Acad Sci U S A. 2004.

Abstract

In eukaryotes, a surveillance mechanism known as nonsense-mediated decay (NMD) degrades the mRNA when a premature-termination codon (PTC) is present. NMD requires translation to read the frame of the mRNA and detect the PTC. During pre-mRNA splicing, the exon-exon junction complex (EJC) is recruited to a region 20-24 nt upstream of the exon junction on the mature mRNA. The presence of a PTC upstream from the EJC elicits NMD. Eukaryotic initiation factor 4A (eIF4A) III is a nuclear protein that interacts physically or functionally with translation initiation factors eIF4G and eIF4B, respectively, and shares strikingly high identity with the initiation factors eIF4AI/II. Here we show that siRNA against eIF4AIII, but not against eIF4AI/II, inhibits NMD. Moreover, eIF4AIII, but not eIF4AI, is specifically recruited to the EJC during splicing. The observations that eIF4AIII is loaded onto the mRNA during splicing in the nucleus, has properties related to a translation initiation factor, and functions in NMD raises the possibility that eIF4AIII substitutes for eIF4AI/II during NMD.

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Figures

Fig. 1.

Fig. 1.

RNAi against eIF4AIII impairs NMD. (A) Northern blot analysis of poly(A) RNA hybridized with Vβ8.1 probe (23). Lanes 1 and 2 represent stable cell lines expressing wild-type TCR-β minigene and PTC-containing TCR-β minigene, respectively. Cycloheximide treatment in both cell lines was performed for 2 h at 37°C (100 μg/ml final) (lanes 3 and 4). siRNAs were transfected into stable cell lines expressing a PTC-containing TCR-β minigene (PTC+) (lanes 5–9) and wild-type TCR-β minigene (PTC–) (lanes 10–14). β-Actin served as a loading marker. The 28S and 18S ribosomal RNA serve to demonstrate equal loading for eIF4AIII siRNA treated samples because siRNA against eIF4AIII is accompanied by decreased levels of actin due to cell death (see Results). CHX, cycloheximide. (B and C) Western analysis of the levels of translation initiation factors after knockdown. Thirty micrograms of protein extract was resolved by SDS/PAGE. Proteins were quantitated against β-actin (Sigma) as a loading control and the level of protein in negative control (4E-T inv) transfected cells was normalized to 100. NT, nontransfected.

Fig. 2.

Fig. 2.

eIF4AIII, but not eIF4AI, associates with the EJC on spliced mRNA. (A) eIF4AIII, but not eIF4AI, is associated with spliced mRNA but not with intronless mRNA (Left). Western analysis showing the presence of eIF4AIII, eIF4AI, and tubulin in the nuclear (NE) and cytoplasmic (Cyto) extracts (Right). (B) eIF4AIII associates with the region of the mRNA that contains the EJC. (Left) Schematic of AdML mRNA showing the locations of oligonucleotides. The numbers indicate the middle of each 12-mer oligonucleotide. (The RNA fragments containing the EJC are designated a, c, and e.) (Right) The total reaction after splicing and oligonucleotide-directed RNase H cleavage was used for the immunoprecipitations and is shown in lanes designated input. Immunoprecipitations were carried out with the indicated antibodies after cleavage with each oligonucleotide. The RNA fragments containing the EJC that were immunoprecipitated are indicated (a, c, and e). The RNA fragments that do not contain the EJC are also indicated (b and d). A darker exposure of lanes 9–12 is included to detect fragment d (55 nt) generated with the +0 oligonucleotide (lane 9). The 13-nt RNA fragment generated with the +36 oligonucleotide is not detected.

Fig. 3.

Fig. 3.

eIF4AIII is associated with spliced mRNA in vivo. (A) Coomassie-stained gel showing GST-eIF4AIII used for microinjection into oocyte cytoplasm. (B) Western analysis of Xenopus oocyte nuclei after preinjection showing the presence of UAP56, a known nuclear protein, and recombinant GST-eIF4AIII. (C) Inj indicates the total RNA before injection, and I indicates the RNA species present in the nuclear lysates before the addition of glutathione beads. Total RNA unbound (U) and bound (B) to the beads is shown.

Fig. 4.

Fig. 4.

eIF4AIII is a shuttling protein. eIF4AIII and 4E-T localization was detected by staining with anti-hemagglutinin antibody (1/1,000 dilution, Covance/Babco) and Texas red-conjugated anti-mouse IgG (Molecular Probes). Human and mouse nuclei are differentiated by staining with Hoechst dye 33258 (Sigma). The mouse nucleus is indicated by an arrow. Leptomycin B (LMB, Sigma) treatments were performed at 5 ng/ml final concentration for 5 h. Images were processed with a Nikon Eclipse E800 microscope at ×60 magnification.

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