Human eIF4AIII interacts with an eIF4G-like partner, NOM1, revealing an evolutionarily conserved function outside the exon junction complex

Abstract

Despite the lack of an exon junction complex (EJC), Saccharomyces cerevisiae contains Fal1p, a DEAD-box helicase highly homologous to eIF4AIII. We show that yeast Fal1p is functionally orthologous to human eIF4AIII, since expression of human eIF4AIII complements both the lethal phenotype and the 18S rRNA biogenesis defect of fal1Δ(null) yeast. We further show that yeast Fal1p interacts genetically with an eIF4G-like protein, Sgd1p: One allele of sgd1 acts as a dominant extragenic suppressor of a mutation in a predicted RNA-binding residue of Fal1p, whereas another synthetically exacerbates the growth defect of this fal1 mutation. Both sgd1 mutations map to a single, short, evolutionarily conserved patch that matches key eIF4A-interacting residues of eIF4G when superimposed on the X-ray structure of the eIF4A/eIF4G complex. We demonstrate direct physical interactions between yeast Sgd1p and Fal1p, and between their human orthologs (NOM1 and eIF4AIII) in vitro and in vivo, identifying human NOM1 as a missing eIF4G-like interacting partner of eIF4AIII. Knockdown of eIF4AIII and NOM1 in human cells demonstrates that this novel conserved eIF4A/eIF4G-like complex acts in pre-rRNA processing, adding to the established functions of eIF4A/eIF4G in translation initiation and of eIF4AIII as the core component of the EJC.

Figure 1.

Human eIF4AIII is homologous to and can replace S. cerevisiae Fal1p in vivo. (A) Amino acid sequence alignment of eIF4AIII homologous proteins from humans, Mus musculus, Xenopus laevis, D. melanogaster, C. elegans, and S. cerevisiae. Walker A and B boxes are shown in gray rectangles. The sequence of D. melanogaster VASA is at the bottom. Also indicated are amino acids in the Walker boxes of human eIF4AIII, in which mutations (K88N and E188Q) were reported previously (Shibuya et al. 2006; Zhang and Krainer 2007) to abolish ATPase activity in vitro, but not NMD in vivo or EJC assembly in vitro (•), and a conserved threonine (T322), whose mutation to valine in S. cerevisiae Fal1p resulted in a CS phenotype (▴). (B) Expression of human or D. melanogaster eIF4AIII complements the lethal phenotype of yeast fal1Δ, as observed by plasmid shuffle (Supplemental Fig. S1A); Walker A and B boxes are required for complementation. (H.eIF4AIII) human eIF4AIII; [H.eIF4AIII(K88N) and H.eIF4AIII(E188Q)] Walker A and B box mutants of human eIF4AIII; (D.eIF4AIII) D. melanogaster eIF4AIII; (Fal1) S. cerevisiae Fal1p; (H.eIF4AI and H.eIF4AII) human eIF4AI and eIF4AII. (C) Expression of human and D. melanogaster eIF4AIII complements the lethal phenotype of yeast fal1Δ, as observed by GAL1 promoter shutoff and activation of expression from a copper-inducible promoter (CUP1) with increasing concentrations of Cu²⁺ (Supplemental Fig. S1C). (H.eIF4AIII) human eIF4AIII; (KT/AP) K88T, A89P; (DE/EL) D188E, E189L; (D.eIF4AIII) D. melanogaster eIF4AIII; (Fal1)S. cerevisiae Fal1p.

Figure 2.

Expression of human eIF4AIII restores 18S rRNA biogenesis in the S. cerevisiae fal1Δ strain. (A) Schematic of rRNA processing in S. cerevisiae. (B) Depletion of Fal1p in yeast S. cerevisiae results in a growth defect in liquid culture. (C) Expression of human eIF4AIII from the GAL1 promoter rescues the growth defect of the yeast fal1Δ strain in liquid culture. (D, top panel) Expression of human eIF4AIII restores 18S rRNA levels in the yeast fal1Δ strain (probes L2 and L9). (Middle panel) Expression of human eIF4AIII partially restores 20S rRNA levels in the yeast fal1Δ strain (probe L3). (Bottom panel) Expression of human eIF4AIII partially relieves accumulation of 23S rRNA in the yeast fal1Δ strain (probe L1). Supplemental Figure S2 shows probes.

Figure 3.

SGD1 interacts genetically with FAL1. (A) Extragenic suppressor sgd1(S340Y) alleviates the growth defect of the CS fal1(T322V) strain in liquid culture (YPD) (strains were exponentially grown at 30°C and shifted to 16°C at time 0). (B) Whereas sgd1(S340Y) suppresses the growth defect of the fal1(T322V) strain at 16°C, a nearby mutation [sgd1(N343Y)] in the same protein results in a growth defect synthetic with fal1(T322V) at 37°C (YPD).

Figure 4.

The suppressor mutation sgd1(S340Y) and synthetic mutation sgd1(N343Y) map to a known conserved 12-amino-acid motif that comprises the largest interface between eIF4G and eIF4A. (A) Amino acid sequence alignment of S. cerevisiae protein Sgd1p with putative Sgd1p-like proteins from humans, M. musculus, D. melanogaster, and C. elegans. Predicted MIF4G and MA3 domains (Ponting 2000) are indicated with blue and purple boxes, respectively. The conserved 12-amino-acid motif (KSLLNKLTLEMF in yeast eIF4G and NSSLNKLSDSNI in yeast Sgd1p), representing the largest surface of eIF4G interacting with eIF4A (Schutz et al. 2008), is indicated with a red box. Mutations in Sgd1p that result in genetic interactions with fal1(T/V) are indicated with triangles; each of these residues is known to form two intermolecular hydrogen bonds with eIF4A, based on the crystal structure of the eIF4G/eIF4A complex (Schutz et al. 2008). (B) Structure of the eIF4G/eIF4A complex (Schutz et al. 2008) showing the locations of residues homologous to the starting mutation in Fal1p (eIF4A) and the suppressor and synthetic mutations in Sgd1p (eIF4G). The major eIF4A/eIF4G interaction interface is marked with a red oval.

Figure 5.

Direct physical interactions between S. cerevisiae Fal1p and Sgd1p and between human eIF4AIII and NOM1. (A) Yeast Sgd1p and Fal1p coimmunoprecipitate. Western blot of IgG precipitates from extracts of formaldehyde cross-linked yeast cells expressing tagged and untagged versions of yeast Sgd1p and Fal1p (Supplemental Fig. S6A), as indicated. All proteins were expressed from their own promoters. A TAP-tagged yeast protein (Trm8p) with known nuclear localization provided a negative control. TAP-tagged proteins were precipitated using bead-immobilized rabbit IgG (the ZZ domain of Staphylococcus aureus protein A present in the TAP tag binds IgG) (Rigaut et al. 1999). The noncovalent complex of horseradish peroxidase (HRP) with polyclonal rabbit anti-peroxidase (PAP) was used for Western blot detection of TAP-tagged proteins (Puig et al. 2001) (the ZZ domain of protein A in the TAP tag binds PAP); detection of the c-myc epitope used HRP-conjugated mouse monoclonal antibody 9E10. (B) Human eIF4AIII and NOM1 coimmunoprecipitate. Western blot of Flag-purified samples from a nuclear extract of human HEK293 cells expressing transiently transfected tagged and untagged versions of human eIF4AIII and NOM1, as indicated (no cross-linking required). Flag-tagged human protein METTL1, with known nuclear localization provided a negative control. (C) Endogenous human eIF4AIII was identified by mass spectrometry among 24 protein bands specifically copurifying from a nuclear extract of human HEK293 cells with Flag-tagged NOM1 (silver staining) (Supplemental Fig. S6B). (D) Direct interaction between heterologously expressed and purified human GST-eIF4AIII and NOM1 is demonstrated by GST pull-down (the gel is Coomassie-stained). The arrow shows the position of full-length NOM1 copurified with GST-eIF4AIII. GST-tagged purified yeast protein Trm82p provided a negative control. (Lane 8) Additional bands in the NOM1 preparation represent N terminally truncated forms of NOM1. I1 and I2 denote inputs for pull-downs shown in lanes 1 and 2, and lanes 3 and 4, respectively.

Figure 6.

The extragenic suppressor mutation sgd1(S340Y) alleviates 18S rRNA biogenesis defects of the CS fal1(T322V) strain; SGD1 is required for rRNA processing at the same sites (A0, A1, and A2) as FAL1. (A) The 18S rRNA biogenesis defect of the S. cerevisiae CS strain fal1(T322V) and its suppression by sgd1(S340Y) are visualized by Northern blot using DNA oligonucleotide probes L2 and L9 (top panel), L1 (middle panel), and L3 (bottom panel); probes are shown in Supplemental Figure S2. Total RNA was prepared from strains grown as in Figure 3A. (B) SGD1 is required for 18S rRNA biogenesis at sites A0, A1, and A2 (same sites as Fal1p). Depletion of P_GAL1-expressed Sgd1p in glucose-containing media at 16°C results in a decrease in 18S rRNA levels (top panel), an increase in 23S rRNA levels (middle panel), a decrease in 20S rRNA levels (bottom panel), and accumulation of the 35S rRNA primary transcript (middle and bottom panels) in the yeast sgd1Δ strain (Northern blot was probed as in A; schematics of the strains are shown in Supplemental Fig. S7A, and growth curves are shown in Supplemental Fig. S7B). Arrows on the left indicate changes in the levels of rRNAs or intermediates in fal1(T/V) (A) and sgd1Δ (B) strains compared with wild type.

Figure 7.

siRNA-mediated knockdown of either eIF4AIII or NOM1 results in an 18S rRNA biogenesis defect in human cells. (A) Schematic of rRNA processing in humans. (B) siRNA-mediated knockdown of eIF4AIII results in a decreased rate of 18S rRNA formation, as determined by in vivo pulse-labeling of HEK293T cells with [methyl-³H] -methionine followed by chase with cold methionine. (Middle panel) Processing of 45S and formation of 41S, 30S/32S, and 21S pre-RNAs are delayed, resulting in a decrease in mature 18S rRNA, suggesting defects at sites 1 and 2. (Right panel) Cotransfection of a plasmid expressing an siRNA-resistant version of eIF4AIII partially rescues the rate of formation of 18S rRNA and precursors, confirming specificity. (C) siRNA-mediated knockdown of NOM1 results in a decreased rate of 18S rRNA formation, as determined by pulse-chase performed as in B. (Middle panel) Formation of 41S and 21S pre-RNAs is delayed, resulting in a decrease in mature 18S rRNA, suggesting a processing defect at site 1. Consistently, a decreased ratio of mature small (18S) to large (28S) subunit rRNA is observed after 3 h of chase (cf. lanes 6 and 12). (Right panel) Cotransfection of a plasmid expressing an siRNA-resistant variant of NOM1 partially rescues the rate of appearance of 18S rRNA as well as 21S and 41S precursors.