Inhibition of nonsense-mediated mRNA decay (NMD) by a new chemical molecule reveals the dynamic of NMD factors in P-bodies

Abstract

In mammals, nonsense-mediated mRNA decay (NMD) is a quality-control mechanism that degrades mRNA harboring a premature termination codon to prevent the synthesis of truncated proteins. To gain insight into the NMD mechanism, we identified NMD inhibitor 1 (NMDI 1) as a small molecule inhibitor of the NMD pathway. We characterized the mode of action of this compound and demonstrated that it acts upstream of hUPF1. NMDI 1 induced the loss of interactions between hSMG5 and hUPF1 and the stabilization of hyperphosphorylated isoforms of hUPF1. Incubation of cells with NMDI 1 allowed us to demonstrate that NMD factors and mRNAs subject to NMD transit through processing bodies (P-bodies), as is the case in yeast. The results suggest a model in which mRNA and NMD factors are sequentially recruited to P-bodies.

Cytoplasmic mRNP granules at a glance

“…More often, 5¢ repression-decay factors interact with mRNA-specific protein complexes that promote translation initiation inhibition, mRNA decay or both. These include the RNA-induced silencing complex (RISC), which silences mRNAs that are targeted by microRNAs (miRNAs) or small interfering RNAs (siRNAs) (Jakymiw et al, 2005;Liu et al, 2005;Pauley et al, 2006); the Upf complex, which targets mRNAs with premature termination codons for nonsense-mediated decay (Durand et al, 2007;Sheth and Parker, 2006); and the related proteins tristetraprolin (TTP) and butyrate response factor 1 (BRF1), which target AU-rich element (ARE)-containing mRNAs for decay (Franks and Lykke-Andersen, 2007). Evidence suggests that RISC-associated GW182 proteins promote RISC-bound mRNP assembly into PBs also independently of 5¢ repression-decay factors (Behm-Ansmant et al, 2006;Eulalio et al, 2009).…”

Processing bodies are not required for mammalian nonsense-mediated mRNA decay

“…The exact mechanism by which SMG5, SMG6, and SMG7 participate in the degradation of the PTC-containing (PTC+) mRNA and where in the cells this happens is still unclear. In human cells, SMG7 was found to localize to distinct cytoplasmic foci called processing bodies (P-bodies, also known as ''GW182-bodies,'' ''DCP1-foci,'' or ''XRN1-foci''), and SMG5 and UPF1 localize to P-bodies when SMG7 is overexpressed (Unterholzner and Izaurralde 2004;Fukuhara et al 2005;Durand et al 2007). In contrast, the metazoan-specific NMD factor SMG6, which initiates degradation of PTC+ mRNA by an endonucleolytic cleavage near the PTC in Drosophila and human cells (Huntzinger et al 2008;Eberle et al 2009), does not colocalize with P-bodies in HeLa cells (Unterholzner and Izaurralde 2004;L Stalder and O Mühlemann, unpubl.).…”

Unraveling regulation and new components of human P-bodies through a protein interaction framework and experimental validation

“…Xrn1), or miRNA-mediated gene silencing (e.g., Ago1-4 and GW182) (Parker and Sheth 2007). Factors involved in nonsense-mediated decay (NMD) were also enriched in P-bodies when NMD was interrupted in yeast (Sheth and Parker 2006) and in mammalian cells (Durand et al 2007). In contrast, most factors involved in mRNA translation (e.g., eIF4G and ribosome subunits) are normally absent from P-bodies (Kedersha et al 2005).…”