Control of mRNA Stability in Fungi by NMD, EJC and CBC Factors Through 3'UTR Introns.

Abstract

In higher eukaryotes the accelerated degradation of mRNAs harboring premature termination codons is controlled by nonsense-mediated mRNA decay (NMD), exon junction complex (EJC), and nuclear cap-binding complex (CBC) factors, but the mechanistic basis for this quality-control system and the specific roles of the individual factors remain unclear. Using Neurospora crassa as a model system, we analyzed the mechanisms by which NMD is induced by spliced 3'-UTR introns or upstream open reading frames and observed that the former requires NMD, EJC, and CBC factors whereas the latter requires only the NMD factors. The transcripts for EJC components eIF4A3 and Y14, and translation termination factor eRF1, contain spliced 3'-UTR introns and each was stabilized in NMD, EJC, and CBC mutants. Reporter mRNAs containing spliced 3'-UTR introns, but not matched intronless controls, were stabilized in these mutants and were enriched in mRNPs immunopurified from wild-type cells with antibody directed against human Y14, demonstrating a direct role for spliced 3'-UTR introns in triggering EJC-mediated NMD. These results demonstrate conclusively that NMD, EJC, and CBC factors have essential roles in controlling mRNA stability and that, based on differential requirements for these factors, there are branched mechanisms for NMD. They demonstrate for the first time autoregulatory control of expression at the level of mRNA stability through the EJC/CBC branch of NMD for EJC core components, eIF4A3 and Y14, and for eRF1, which recognizes termination codons. Finally, these results show that EJC-mediated NMD occurs in fungi and thus is an evolutionarily conserved quality-control mechanism.