Two mammalian MAGOH genes contribute to exon junction complex composition and nonsense-mediated decay

Abstract

The exon junction complex (EJC) participates in the regulation of many post-transcriptional steps of gene expression. EJCs are deposited on messenger RNAs (mRNAs) during splicing and their core consists of eIF4A3, MLN51, Y14, and MAGOH. Here, we show that two genes encoding MAGOH paralogs (referred to as MAGOH and MAGOHB) are expressed in mammals. In macrophages, the expression of MAGOHB, but not MAGOH mRNA, increases rapidly after LPS stimulation. Both MAGOH proteins interact with other EJC components, incorporate into mRNA-bound EJCs, and activate nonsense-mediated decay. Furthermore, the simultaneous depletion of MAGOH and MAGOHB, but not individual depletions, impair nonsense-mediated decay in human cells. Hence, our results establish that the core composition of mammalian EJCs is more complex than previously recognized.

Keywords: EJC; MAGOHB; NMD; gene duplication; mago nashi homolog.

Figure 4. Specific knockdown of MAGOHB and MAGOH. (A) HeLa cells were transfected with siRNAs targeting MAGOH (siRNAs 1, 2, and 3), MAGOHB (siRNAs 4, 5, and 6) or both MAGOH and MAGOHB (siRNA combinations 7, 8, and 9, see Materials and Methods). Expression levels of MAGOH or MAGOHB mRNAs were determined by quantitative RT-PCR and normalized to TATA binding protein (TBP). Reduction of mRNA levels of MAGOH and MAGOHB compared with Luc-siRNA transfected cells (100%) is depicted; data represent the mean percentage (± standard deviation) from three independent experimets. (B) Western blot of HeLa cells that were transfected with the indicated siRNA or siRNA combinations. The knockdown efficiency was assessed with MAGOH-specific antibodies.

Figure 1. Alignment and conservation of the two MAGOH genes/proteins. (A) Human MAGOHB and MAGOH gene structures, exons, and introns are shown as boxes and lines, respectively. The sizes of exons and introns in base pairs (bp) are depicted above and below the diagram, respectively. (B) Maximum likelihood phylogeny estimated with the PhyML algorithm using the ORF sequences of MAGOH and MAGOHB from Homo sapiens (HS), Mus musculus (MM), Bos taurus (BT), Canis lupus (CL), and Cricetulus griseus (CG). The scale bar represents the measure of phylogenetic distance and corresponds to four base substitutions per 100 positions. Numbers at nodes represent bootstrap values. See also Figure S1C. Alignment of the two human MAGOH proteins.

Figure 2. Expression of MAGOH and MAGOHB transcripts. (A) Quantitative RT-PCR analysis of endogenous MAGOHB and MAGOH transcripts in HEK293 and HeLa cells. The expression levels were normalized to TATA binding protein (TBP). (B) Quantitative RT-PCR analysis of the MAGOHB and MAGOH transcripts levels in various tissues of three adult mice (data of individual mice are shown in Fig. S2). (C) Quantitative RT-PCR analysis of the MAGOHB and MAGOH transcripts levels in murine derived macrophages that were stimulated with LPS (10 ng/mL) for 0, 1, 6, 12, and 24 h. Data presented in (A–C) represent the mean value (± standard deviation) obtained from three independent experiments. (D) Western blot of macrophages that were stimulated with LPS (10 ng/mL) for 0, 1, 6, 12, and 24 h. MAGOH protein levels were assessed with MAGOH-specific antibodies.

Figure 3. MAGOHB and MAGOH interact with the same proteins and elicit NMD. (A) FLAG-MAGOH and FLAG-MAGOHB were expressed in HeLa cells. Complexes were immunoprecipitated using FLAG-agarose beads. Western blotting with specified antibodies was used to visualize co-precipitated endogenous proteins. (B) Equal amounts of FLAG-Y14 were transfected together with increasing amounts of V5-MAGOHB or V5-MAGOH. Immunoprecipitations were performed using FLAG beads and co-precipitated components of the EJC were detected by immunoblotting. (C) Equal amounts of FLAG-MAGOH and FLAG-MAGOHB were transfected together with increasing amounts of V5-MAGOHB and V5-MAGOH respectively. Immunoprecipitations were performed using FLAG beads and immunoblotting was performed to visualize co-precipitated proteins. (D) A ³²P bodylabeled splicing substrate (MINX RNA) was incubated in HeLa nuclear extracts and HEK293 whole cell extracts expressing FLAG-MAGOHB, FLAG-MAGOH, or FLAG-Y14. FLAG-tagged proteins were immunoprecipitated and co-precipitated RNA was separated by denaturing PAGE. (E) Northern blot of HeLa cells co-transfected with the β-globin 4boxB reporter plasmid, a control and the indicated λNV5-fusion proteins. Percentages represent the mean calculated from three independent experiments.

Figure 5. MAGOH and MAGOHB collectively support NMD. (A and B) The effect of single or combined knockdowns of MAGOH and MAGOHB on NMD was determined. The levels of the known cellular NMD-substrates SC35 1.6 kb (A) SC35 1.7 kb (B) were measured by quantitative RT-PCR and normalized to TBP. Fold upmodulation compared with Luc-siRNA transfected cells is depicted. Values for Y14-depleted cells are shown as separate panel. (C) Both MAGOH and MAGOHB are incorporated into the EJC core. Hence, two different types of core EJCs (MAGOH-containing and MAGOHB-containing) are formed in mammalian cells.