A Competition between Stimulators and Antagonists of Upf Complex Recruitment Governs Human Nonsense-Mediated mRNA Decay

Abstract

The nonsense-mediated decay (NMD) pathway subjects mRNAs with premature termination codons (PTCs) to rapid decay. The conserved Upf1–3 complex interacts with the eukaryotic translation release factors, eRF3 and eRF1, and triggers NMD when translation termination takes place at a PTC. Contrasting models postulate central roles in PTC-recognition for the exon junction complex in mammals versus the cytoplasmic poly(A)-binding protein (PABP) in other eukaryotes. Here we present evidence for a unified model for NMD, in which PTC recognition in human cells is mediated by a competition between 3′ UTR–associated factors that stimulate or antagonize recruitment of the Upf complex to the terminating ribosome. We identify cytoplasmic PABP as a human NMD antagonizing factor, which inhibits the interaction between eRF3 and Upf1 in vitro and prevents NMD in cells when positioned in proximity to the termination codon. Surprisingly, only when an extended 3′ UTR places cytoplasmic PABP distally to the termination codon does a downstream exon junction complex enhance NMD, likely through increasing the affinity of Upf proteins for the 3′ UTR. Interestingly, while an artificial 3′ UTR of >420 nucleotides triggers NMD, a large subset of human mRNAs contain longer 3′ UTRs but evade NMD. We speculate that these have evolved to concentrate NMD-inhibiting factors, such as PABP, in spatial proximity of the termination codon.

Autoregulation of the nonsense-mediated mRNA decay pathway in human cells

“…4B). Similar to UPF1 and SMG7 and consistent with the findings by Singh et al (2008), the 39 UTR of SMG5 significantly reduced mini mu reporter mRNA levels, and NMD abrogation led to a fourfold to sixfold increase of transcript levels (Fig. 5C).…”

“…Data from different laboratories indicate that a 39 UTR of >420 nt can trigger NMD (Eberle et al 2008;Singh et al 2008). However, the majority of the z55% of human mRNAs with a 39 UTR of >500 nt (Supplemental Table III) are obviously not NMD substrates.…”

“…When the ribosome reaches a TC located in a messenger ribonucleoprotein (mRNP) environment where it fails to receive the PABP-mediated termination-stimulating signal, the ribosome stalls for a prolonged period of time, allowing binding of UPF1, a key factor in NMD, to eRF3 (Singh et al 2008). This event marks the mRNA for degradation; however, at this point the commitment to the NMD pathway is thought to still be reversible.…”

“…The experimental evidence from several different model organisms suggests, for example, that long 39 UTRs can elicit NMD (Muhlrad and Parker 1999;Amrani et al 2004;Kertesz et al 2006;Behm-Ansmant et al 2007;Longman et al 2007;Eberle et al 2008;Kerenyi et al 2008;Singh et al 2008;Hansen et al 2009;Kebaara and Atkin 2009). A greater distance between the TC and PABP would impede transmission of the PABP-mediated termination-stimulating signal to the ribosome stalled at the TC, thus tilting the balance from efficient termination and ribosome release toward NMD.…”

Introns in UTRs: Why we should stop ignoring them

“…Current models suggest that the mRNA is translated only once before it is destroyed, producing a single molecule of protein per transcript [37]. Upf1 and Upf2 can also interact on a transcript and stimulate NMD when the transcript has a particularly long 3 0 -UTR [38]. This additional form of NMD is presumably splicing-independent, and is discussed elsewhere [7,38].…”