Splicing remodels messenger ribonucleoprotein architecture via eIF4A3-dependent and -independent recruitment of exon junction complex components

Abstract

Pre-mRNA splicing not only removes introns and joins exons to generate spliced mRNA but also results in remodeling of the spliced messenger ribonucleoprotein, influencing various downstream events. This remodeling includes the loading of an exon-exon junction complex (EJC). It is unclear how the spliceosome recruits the EJC onto the mRNA and whether EJC formation or EJC components are required for pre-mRNA splicing. Here we immunodepleted the EJC core component eIF4A3 from HeLa cell nuclear extract and found that eIF4A3 is dispensable for pre-mRNA splicing in vitro. However, eIF4A3 is required for the splicing-dependent loading of the Y14/Magoh heterodimer onto mRNA, and this activity of human eIF4A3 is also present in the Drosophila ortholog. Surprisingly, the loading of six other EJC components was not affected by eIF4A3 depletion, suggesting that their binding to mRNA involves different or redundant pathways. Finally, we found that the assembly of the EJC onto mRNA occurs at the late stages of the splicing reaction and requires the second-step splicing and mRNA-release factor HRH1/hPrp22. The EJC-dependent and -independent recruitment of RNA-binding proteins onto mRNA suggests a role for the EJC in messenger ribonucleoprotein remodeling involving interactions with other proteins already bound to the pre-mRNA, which has implications for nonsense-mediated mRNA decay and other mRNA transactions.

Fig. 1.

eIF4A3 is dispensable for pre-mRNA splicing in vitro. (A) Endogenous eIF4A3 is efficiently immunodepleted from HeLa NE. eIF4A3 and the indicated control proteins were detected by Western blotting. AK105 is a control monoclonal antibody against bacterial maltose-binding protein. (B) The EJC component Y14 and the small nuclear RNP protein U2-B″ are not codepleted with eIF4A3 under these conditions. (C) In vitro splicing time course with human β-globin pre-mRNA in untreated NE (lanes 1–4), mock-depleted NE (lanes 5–8), and eIF4A3-depleted NE (Δ4A3 NE) without (lanes 9–12) or with (lanes 13–16) added recombinant His-tagged eIF4A3 (80 ng). The pre-mRNA, mRNA, and intermediates are indicated. (D) In vitro splicing assays with β-globin, IgM C3-C4, Ftz, and AdML pre-mRNAs in mock-depleted and Δ4A3 NE. The arrowheads show the spliced mRNAs.

Fig. 2.

eIF4A3 is essential for loading Y14/Magoh and for EJC core assembly on spliced mRNA. (A) Δ4A3 NE cannot load Y14/Magoh onto mRNA during splicing. GST pull-down assays were performed after in vitro splicing of β-globin pre-mRNA using mock-depleted or Δ4A3 NE. The input and pull-down samples are shown: lanes 1–4, GST alone; lanes 5–8, GST-Y14/His-Magoh; lanes 9–12, GST-eIF4A3. (B) Adding back recombinant eIF4A3 to Δ4A3 NE fully restores loading of Y14/Magoh onto spliced mRNA. GST-Y14/His-Magoh pull-down was carried out from mock-depleted NE (lane 11) and from Δ4A3 NE (lanes 12–16). Purified recombinant His-tagged eIF4A3 (10, 20, 50, 100, and 200 ng) was added to reactions containing 20 μl of Δ4A3 NE (lanes 12–16). A total of 20 μl of untreated NE has ≈400 ng of eIF4A3. (C) Comparison of different eIF4A3 mutants or the Drosophila ortholog (200 ng each) in restoring the Y14/Magoh-loading activity to Δ4A3 NE. The mRNA pull-down efficiency is indicated below the relevant lanes.

Fig. 3.

Other EJC-associated factors bind to mRNA independently of eIF4A3. (A) eIF4A3 is not required for binding of MLN51 to mRNA but enhances its association with mRNA after splicing. GST pull-down assays were carried out from splicing reactions with β-globin pre-mRNA in the presence of GST–SELOR, GST-Y14/His-Magoh (GY/HM), or GST-eIF4A3 in either mock-depleted NE (lanes 1–12) or ΔeIF4A3 NE (lanes 13–24). After washing with 150 mM KCl buffer (lanes 5–8 and 17–20) or 500 mM KCl buffer (lanes 9–12 and 21–24), the bound RNA was recovered and analyzed by denaturing PAGE and autoradiography. Loading of UAP56 and Aly/REF (B), RNPS1 (C), and Upf3b and Upf3a (D) onto mRNA during splicing is not affected by depletion of eIF4A3. All of the tested proteins were GST-tagged. Fifteen percent to 20% of each reaction was loaded in the input lanes.

Fig. 4.

Loading of eIF4A3 and assembly of the EJC core on mRNA occur at the late stages of splicing and require the helicase activity of HRH1/Prp22. (A) In vitro splicing and GST pull-down using WT (WT-AG; lanes 1–12) and 3′ splice site mutant (Mut-GG; lanes 13–24) pre-mRNAs. The GST fusion proteins added to each reaction are indicated above each lane. The mobilities of the splicing precursor, intermediates, and products are shown on the right. The Mut-GG substrate can undergo only the first step of splicing. (B) In vitro splicing and GST pull-down in the presence of WT (lanes 1–6) or LAT-mutant HRH1 (lanes 7–12) (200 ng). The mRNA pull-down efficiency is indicated below the gel. (C) Purification of in vitro-assembled mRNPs after splicing using β-globin pre-mRNA with two MS2-binding sites at the 3′ end of exon 2. Splicing reactions were incubated with GST-MS2 in the presence of WT or LAT HRH1 (lanes 1 and 2). After 3 h, DNA oligonucleotides complementary to the intron were added, and incubation was continued for 20 min (lanes 3 and 4). mRNPs were purified on glutathione-Sepharose, and bound RNAs were extracted and analyzed by denaturing PAGE (lanes 5 and 6). The purified 3′-cleaved products from pre-mRNA are also labeled (lanes 5 and 6). (D) Proteins from the purified mRNPs from B (lanes 5 and 6) were analyzed by Western blotting using antibodies against eIF4A3 and Aly/REF.