Eukaryotic gene expression consists of a series of events mediating the information flow from DNA via mRNA to protein. Cellular surveillance mechanisms exist to detect and eliminate erroneous mRNA in order to prevent the production of incorrect transcripts. Nonsense-mediated mRNA decay (NMD) targets mRNA for degradation, which terminate translation prematurely or incorrectly. Thereby, NMD prevents the synthesis of unfunctional or harmful peptides. Besides this quality control function, NMD also regulates the levels of many full-length protein encoding mRNA.
The messenger ribonucleoprotein (mRNP) architecture downstream of the stop codon is the main determinant for the initiation of the NMD pathway. Exon-junction complexes (EJCs) and long 3′ untranslated regions (UTRs) are known stimulators of NMD. EJCs are central components of the gene expression pathway and are deposited upon splicing on the mRNA. The exact mechanism how these mRNP elements induce NMD is unclear. Moreover, the series of molecular events ultimately leading to the degradation of the mRNA, as well as the precise interplay of NMD factors during this process, are not well defined.
In this cumulative work, several important steps in the NMD pathway were investigated. I could show that that for NMD suppression, an interaction cascade involving the eukaryotic release factor 3 (eRF3), the cytoplasmic poly(A) binding protein (PABPC1) and the cap-binding EIF4F complex component eIF4G is required. This suggests that efficient ribosome recycling is important for the normal termination of translation, which in turn prohibits the activation of NMD. To gain insight into the mode of EJC assembly during splicing, CWC22, an essential splicing component, was identified as the critical EJC loading factor. Remarkable differences were observed when comparing long 3′ UTRs and EJCs as NMD-inducing elements. These differences involved not only the efficiency of mRNA degradation, the mode of NMD activation, but also the requirements of NMD factors. These results indicate that EJCs are highly evolved mRNP markers, which utilize a specific mechanism to achieve efficient degradation of the target mRNA. In contrast, long 3′ UTRs influence the mRNP composition around the stop codon, thus impairing regular translation termination and leading to infrequent and less efficient mRNA degradation. In conclusion, this work illuminates multiple aspects of mammalian NMD and highlights the important missing pieces of information, which are to be uncovered by future research
