Gene expression in eukaryotes involves multiple post-transcriptional steps, including pre–messenger RNA (mRNA) processing, the export of the mature mRNA to the cytoplasm, its correct intracellular localization, and finally its translation and turnover 
. All these processes are coordinated by a network of communicating cellular machines 
. The exon junction complex (EJC) plays a central role in the coordination of post-transcriptional gene expression in metazoan cells. The EJC is deposited on nascent mRNAs during splicing in a sequence-independent manner 20–24 nucleotides (nts) upstream of exon–exon junctions 
. EJCs communicate the pre-splicing architecture of a spliced mRNA to cytoplasmic processes and modulate central events in gene expression such as nuclear mRNA export, mRNA quality control by nonsense-mediated mRNA decay (NMD), and translation of mRNAs in the cytoplasm 
NMD represents an intensively studied splicing- and translation-dependent process that limits the expression of abnormal transcripts containing premature termination codons and controls the expression of normal mRNA isoforms that are generated from the same pre-mRNA at different times of development and in different tissues 
. As such, NMD has broad biological and medical implications 
. NMD can be recapitulated by introducing a functional intron into the 3′ untranslated region (UTR) of an otherwise wild-type mRNA or by tethering either of the EJC components MAGOH, Y14, eIF4A3 (DDX48), or Barentsz (BTZ, also referred to as MLN51 or CASC3) to the 3′ UTR of reporter mRNAs in human cells 
. These data indicate that the presence of an EJC at an appropriate distance downstream of a termination codon is sufficient to elicit NMD and suggest that the EJC provides the direct molecular link for the recognition of premature translation termination codons.
The core of the EJC, consisting of the four proteins eIF4A3, MAGOH, Y14, and BTZ, can be assembled from recombinant subunits in vitro when its components are simultaneously present 
. Such in vitro assembly of the EJC core also requires the presence of ATP and single-stranded RNA, both of which are an integral part of the complex 
. The crystal structure of this core EJC bound to oligo-U RNA shows that eIF4A3 binds the phosphate–sugar backbone of the RNA via its DEAD-box helicase domain. This structure explains why the binding of RNA by the EJC is stable and specific for RNA, despite being sequence-independent 
. Binding of RNA requires simultaneous binding of a molecule of ATP, whereas ATP hydrolysis by eIF4A3's inherent ATPase activity leads to the dissociation of the EJC from the RNA 
. To stably clamp the EJC on the RNA, the ATPase activity of eIF4A3 is inhibited by the binding of the MAGOH-Y14 heterodimer to eIF4A3 
. Interestingly, eIF4A3 can undergo a remarkable structural reorganization 
. Whereas BTZ binds eIF4A3 in both the open and the closed RNA-bound conformations, MAGOH-Y14 binding to eIF4A3 occurs only in its RNA-bound state.
The protein PYM also plays an important role both in the function and in the recycling of EJCs. Ribosome-associated PYM binds to the EJC components MAGOH-Y14 
, thereby recruiting ribosomes to spliced mRNAs to stimulate translation as well as directing the disassembly of EJCs in the cytoplasm 
The determination of the crystal structure of the biochemically assembled EJC core has been a milestone in the analysis of EJC function. However, a better biological understanding of EJC assembly will have to integrate this structural information with the fact that EJC assembly in vivo is strictly splicing-dependent 
. We have thus investigated spliceosome-dependent EJC assembly and demonstrate that the EJC is assembled along a defined hierarchical pathway that ensures the proper positioning and stable binding of the EJC on the (pre-)mRNA substrate. Our data define a stable minimal pre-EJC core consisting of eIF4A3 and MAGOH-Y14, which serves as a binding platform for EJC binding factors like BTZ and UPF3b that link the EJC to functional downstream effectors.