Leveraging Rules of Nonsense-Mediated mRNA Decay for Genome Engineering and Personalized Medicine

Abstract

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA quality control and regulatory process that plays direct roles in human health and disease. In this Minireview, we discuss how understanding the molecular events that trigger NMD can facilitate strategic targeting of genes via CRISPR/Cas9 technologies and also inform disease diagnostics and treatments.

Figure 1. Understanding NMD Can Improve Generating CRISPR/Cas9-Mediated Gene Knockouts

(A) Cytoplasmic mRNA bound by the cap (m⁷G)-binding protein complex (CBC) and poly(A)-binding proteins (PABPs) undergoes a pioneer round of translation coupled to inspection by the NMD machinery (Maquat et al., 2010). When the translating ribosome undergoes an altered termination event at the premature termination codon (PTC) (for example, as a result of indel formation), the SMG1 complex (SMG1C) joins factors that also typify normal termination events, namely eukaryotic release factor 1 (eRF1) and eRF3, to form the SURF complex. SMG1C is composed of the UPF1 kinase SMG1 and its repressors SMG8 and SMG9. EJCs typically display UPF2 anchored by UPF3X. Dashed lines signify that CBC promotes SURF formation and that CBC and PABPs may “circularize” the mRNA via bridging proteins. Notably, PABPs are known antagonists of NMD. AUG, initiation codon; Norm Ter, normal termination codon; nts, nucleotides. (B) As a rule, if an EJC-associated exon-exon junction (E–E) resides ≥50–55 nt downstream of the termination event, then NMD initiates. UPF2 interacts with UPF1, causing a large conformational change that activates UPF1 helicase activity. Within the resulting decay-inducing complex (DECID), SMG1 phosphorylates UPF1. CBC also promotes SMG1-UPF1 binding to the EJC (dashed arrow), and phosphorylated UPF1 inhibits further translation initiation at the AUG codon (dashed arrow). (C) Phosphorylated UPF1 recruits the SMG6 endonuclease, which cleaves between the PTC and EJC, and SMG5-SMG7 recruits the multisubunit CCR4-NOT complex, causing deadenylation, and/or DCP2-DCP1a, causing decapping. In all cases, destruction of mRNA fragments ensues via general exoribonucleases. (D) Examples of indel/PTC placement within a typical transcript and its result on NMD as well as additional scenarios. Y, yes NMD occurs; N, no NMD. See text for details.