Inhibition of Nonsense-mediated mRNA Decay by the Natural Product Pateamine A through Eukaryotic Initiation Factor 4AIII

Abstract

Nonsense-mediated mRNA decay (NMD) in mammalian cells is a key mechanism for the removal of mRNA containing premature stop codons and is mediated by the coordinated function of numerous proteins that dynamically associate with the exon junction complex. The information communicated by these interactions and the functional consequences from a mechanistic perspective, however, are not completely documented. Herein, we report that the natural product pateamine A (PatA) is capable of inhibiting NMD through direct interaction with eIF4AIII, which is independent of its inhibition of translation initiation. Furthermore, we have characterized the mechanisms by which PatA and cycloheximide modulate NMD. Unlike CHX, PatA was found to inhibit NMD by a novel mechanism that is independent of the phosphorylation of Up-frameshift protein 1.In mammalian cells, nonsense-mediated mRNA decay (NMD) 2 is one of the key RNA surveillance mechanisms to specifically degrade mRNA with premature stop codons (PTCs) located more than 50 -55 nucleotides upstream of the final exon-exon junction. PTCs can be formed in genes containing a nonsense mutation or frameshift mutation or as a result of errors that occur during transcription or RNA splicing (1-4). After splicing, the exon junction complex (EJC) imprints mature mRNAs 20 -24 nucleotides upstream of the exon-exon junction (5). The EJC is a dynamic multiprotein complex that plays an essential role in NMD. The core EJC proteins eIF4AIII, Y14, Magoh, and MLN51 form a platform to interact with several other proteins in a dynamic fashion to regulate NMD (6). The spatial-temporal regulation of NMD by the EJC and its partner proteins has been extensively investigated, leading to the proposition of the "linear interaction model" (4, 7). According to this model, deposition of the EJC onto mRNA causes the Y14-Magoh and eIF4AIII complex to effectively recruit Upf3 that interacts with Upf2. Although the mechanism of loading Upf1 onto EJC is poorly understood, it has been shown that phosphorylation and dephosphorylation of Upf1 by SMG-1, -5, -6, and -7 affect the interaction with the EJC complex through

The sld Genetic Defect

“…Retention of intron 54 and subsequent correct splicing of downstream exons will result in transcripts with multiple premature termination codons, prime substrates for degradation by NMD (25). We demonstrate that pateamine A markedly increases aberrant transcripts in mutant glands, indicating that the aberrant message is degraded, consistent with a role for NMD in message degradation (26). These results further indicate that steady-state levels of aberrant transcripts detected by RT-PCR underestimate the proportion of aberrantly spliced transcripts compared with those spliced correctly.…”

“…We therefore hypothesized that inhibition of NMD would result in increased levels of aberrant message with little change in correctly spliced Muc19 mRNA. To test this hypothesis, we incubated minced fragments from 8-week-old sld sublingual glands with pateamine A, a selective inhibitor of NMD through its direct interaction and stimulation of eIF4AIII, one of the core proteins of the exon junction complex, and indirectly via inhibition of eIF4AI/II-mediated translation initiation (26). As shown in Fig.…”

From unwinding to clamping — the DEAD box RNA helicase family

“…Hippuristanol is highly specific: it inhibits ATP binding to eIF4A but does not affect the eIF4AIII homologue. By contrast, pateamine A stimulates the RNA-stimulated ATPase activity of eIF4A but also seems to be active on eIF4AIII 108 . Similarly to the function of pateamine A, the non-coding RNA BC1 binds specifically to eIF4A and stimulates its ATPase activity but blocks its unwinding activity 109 to prevent translation initiation in neurons.…”

Nonsense-mediated mRNA decay in humans at a glance

“…In contrast to Ataluren, cardiac glycosides, which elevate the level of intracellular Ca 2+ , are capable of inhibiting NMD at concentrations that reportedly do not affect cellular viability (Nickless et al, 2014). It might be possible to concomitantly use Ataluren and cardiac glycosides, or possibly a clinically effective nonsense suppressor with either another small reagent that has been reported to inhibit NMD (Bhuvanagiri et al, 2014;Martin et al, 2014;Dang et al, 2009;Feng et al, 2015;Gopalsamy et al, 2012;Usuki et al, 2004) or one or more antisense oligonucleotides that occlude deposition of the one or more EJCs that would normally reside downstream of a particular PTC (Nomakuchi et al, in press). Another approach that might be worthwhile for obtaining full-length protein from diseaseassociated mRNAs is site-directed pseudouridylation of in-frame PTCs (Karijolich and Yu, 2011); by providing a gene-specific therapeutic strategy, such a strategy would be expected to have only minimal toxic side effects.…”