Abstract
Author SummaryThe first step in the expression of eukaryotic protein-coding genes is transcription into a messenger RNA (mRNA) precursor in the nucleus. These precursor mRNAs then undergo maturation through the removal of introns in a process termed splicing. During splicing, the splicing machinery or “spliceosome” deposits a complex of proteins onto the mRNA that accompanies it during post-transcriptional steps in gene expression, including the regulation of mRNA stability, transport out of the nucleus, cellular localisation, and translation. This complex, the exon junction complex (EJC), represents a molecular memory of the splicing process. Understanding the biogenesis of EJCs and their downstream effects helps reveal the basic principles by which the primary steps of mRNA synthesis are coupled to the regulation of gene expression. Here we show that EJCs are assembled in a strictly splicing-dependent manner through an unexpected, coordinated, and hierarchical assembly pathway. Importantly, we show that the EJC recruits the cytoplasmic protein BTZ, which then bridges the complex to an mRNA quality-control machinery called the nonsense-mediated decay pathway that degrades mRNAs containing premature stop codons. This finding suggests that the EJC and bridging by BTZ help determine the stability of mRNA and thus are essential for proper cellular surveillance of mRNA quality.
Highlights
Gene expression in eukaryotes involves multiple post-transcriptional steps, including pre–messenger RNA processing, the export of the mature mRNA to the cytoplasm, its correct intracellular localization, and its translation and turnover [1,2]
The binding of eIF4A3 to BTZ or MAGOH-Y14 can be separated and occur independently from one another. These results indicate that UPF3b binding to the exon junction complex (EJC) requires a heterotrimer consisting of eIF4A3, MAGOH, and Y14 and is likely supported by BTZ
The core EJC consisting of the recombinant proteins eIF4A3, BTZ, MAGOH, and Y14 self-assembles in the presence of ATP on a RNA substrate in vitro [18], but this core EJC does not convey positional information that results from spliceosomal EJC assembly
Summary
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 its translation and turnover [1,2]. NMD can be recapitulated by introducing a functional intron into the 39 untranslated region (UTR) of an otherwise wild-type mRNA or by tethering either of the EJC components MAGOH, Y14, eIF4A3 (DDX48), or Barentsz (BTZ, referred to as MLN51 or CASC3) to the 39 UTR of reporter mRNAs in human cells [13,14,15,16,17]. 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
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