Introns play an essential role in splicing-dependent formation of the exon junction complex

Abstract

Pre-mRNA splicing specifically deposits the exon junction complex (EJC) onto spliced mRNA, which is important for downstream events. Here, we show that EJC components are primarily recruited to the spliceosome by association with the intron via the intron-binding protein, IBP160. This initial association of EJC components occurs in the absence of the final EJC-binding site on the exon. RNA interference (RNAi) knockdown of IBP160 arrested EJC association with cytoplasmic RNAs following nonsense-mediated decay. We propose that the intron has a crucial role in the early steps of EJC formation and is indispensable for the subsequent formation of a functional EJC.

Figure 1.

Interaction of EJC components with intron-bound IBP160 in the spliceosomal C1 complex. (A) The experimental design for coimmunoprecipitation of UV-cross-linked IBP160. The ³²P-labeled site, located 40 nt upstream of the branchpoint site (BPS) in Adv pre-mRNA, is indicated by an asterisk. The 3′ splice site substituted with GG is shown. The hexagon (black), the smaller oval (dark gray), and the larger oval (light gray) represent IBP160, the IBP160 subcomplex, and the whole spliceosome, respectively. (B,C) Coimmunoprecipitation of the cross-linked IBP160 with antibodies against eIF4AIII, RNPS1, or SRm160 (B), or Flag tag (C). Aliquots of the total samples (10%) were loaded in the input lanes. The EJC component expressed in each HEK293 nuclear extract used is indicated above each lane in C. (D) Size fractionation of the IBP160 subcomplex by gel filtration chromatography (Sephacryl S500HR column). The numbers to the left of the panel represent the molecular weight markers. (E) Coimmunoprecipitation with Flag-eIF4AIII mutants. The mutated motifs are shown above each lane. The arrowheads show the ³²P-labeled cross-linked IBP160 in B–E.

Figure 2.

Association of EJC components with the C1 complex in the absence of the final EJC-binding site in the upstream exon. (A) The two shortened pre-mRNA splicing substrates are shown schematically. The lengths of the upstream exons are shown on the left. The labeled nucleotide and the 3′ splice site mutation are shown as in Figure 1A. (B) In vitro splicing of 17GG and 38GG pre-mRNAs. The identities of the RNA species are shown on the right. (C) Coimmunoprecipitation of IBP160 cross-linked either to 38GG or to 17GG, indicated as 38 or 17 above each lane, with the αFlag antibody as in Figure 1C.

Figure 3.

IBP160 is required for efficient EJC association and NMD. (A) Western blot to examine the effects of RNAi. The siRNAs used are indicated above the panels. The antibodies used are shown on the left. (B) Quantitative RT–PCR to measure the levels of two ncRNAs (Gas5 and UHG) and two control mRNAs (Actin and GAPDH) in siRNA-treated cells. The bottom panel shows quantitation of the cytoplasmic RNAs. (C) Northern blot analysis with the RNAs used in B. The slowly migrating bands in the UHG panel are likely to be the splicing intermediates containing certain intron(s). (D) Immunoprecipitation of two ncRNAs (Gas5 and UHG) associated with EJC in the cytoplasm of HeLa cells. The RNA levels immunoprecipitated with αPABP1, αeIF4AIII, and αY14 were measured by quantitative RT–PCR. The ratios of immunoprecipitated RNA efficiencies between control and ΔIBP160 cells were calculated from three independent experiments and graphed. The immunoprecipitation (IP) efficiency is represented as the ratio (percentage) between RNA amounts in immunoprecipitates and input (IP efficiency = RNA amounts in immunoprecipitates/input × 100). The immunoprecipitation efficiency of each antibody from the control cells is as follows: αPABP1, 1.4% for Gas5 and 1.6% for UHG; αeIF4AIII, 0.02% for Gas5 and 0.15% for UHG; αY14, 0.75% for Gas5 and 4.2% for UHG. The statistical analysis of the results between αPABP1 and either αeIF4AIII or αY14 were examined by t-test; each P-value is indicated above the bar.

Figure 4.

Model of EJC formation during pre-mRNA splicing. The white ovals represent EJC components. Other symbols are as shown in Figure 1A.