Regulation of nonsense-mediated mRNA decay: Implications for physiology and disease

Nonsense-mediated mRNA decay (NMD) is an mRNA quality control mechanism that destabilizes aberrant mRNAs harboring premature termination (nonsense) codons (PTCs). Recent studies have shown that NMD also targets mRNAs transcribed from a large subset of wild-type genes. This raises the possibility that NMD itself is under regulatory control. Indeed, several recent studies have shown that NMD activity is modulated in specific cell types and that key components of the NMD pathway are regulated by several pathways, including microRNA circuits and NMD itself. Cellular stress also modulates the magnitude of NMD by mechanisms that are beginning to be understood. Here, we review the evidence that NMD is regulated and discuss the physiological role for this regulation. We propose that the efficiency of NMD is altered in some cellular contexts to regulate normal biological events. In disease states—such as in cancer—NMD is disturbed by intrinsic and extrinsic factors, resulting in altered levels of crucial NMD-targeted mRNAs that lead to downstream pathological consequences. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Mechanism and regulation of the nonsense-mediated decay pathway

The Nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs) but also regulates the abundance of a large number of cellular RNAs. The central role of NMD in the control of gene expression requires the existence of buffering mechanisms that tightly regulate the magnitude of this pathway. Here, we will focus on the mechanism of NMD with an emphasis on the role of RNA helicases in the transition from NMD complexes that recognize a PTC to those that promote mRNA decay. We will also review recent strategies aimed at uncovering novel trans-acting factors and their functional role in the NMD pathway. Finally, we will describe recent progress in the study of the physiological role of the NMD response.     

Integration of splicing, transport and translation to achieve mRNA quality control by the nonsense-mediated decay pathway

When pre-mRNAs are spliced, a multi-component complex is deposited onto them, close to the sites of intron removal. New findings suggest that these exon-exon junction complexes and the complexes that bind mRNA caps are key effectors of the fate of spliced mRNAs and may regulate whether mRNAs containing premature stop codons are degraded.     

Caspases shutdown nonsense-mediated mRNA decay during apoptosis

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that plays integral roles in eliminating mRNAs with premature termination codons to prevent the synthesis of truncated proteins that could be pathogenic. One response to the accumulation of detrimental proteins is apoptosis, which involves the activation of enzymatic pathways leading to protein and nucleic acid cleavage and culminating in cell death. It is not clear whether NMD is required to ensure the accurate expression of apoptosis genes or is no longer necessary since cytotoxic proteins are not an issue during cell death. The present study shows that caspases cleave the two NMD factors UPF1 and UPF2 during apoptosis impairing NMD. Our results demonstrate a new regulatory pathway for NMD that occurs during apoptosis and provide evidence for role of the UPF cleaved fragments in apoptosis and NMD inhibition.Cell death is a natural process that occurs throughout development and the life of a multicellular organism removing cells that are no longer needed or have become pathogenic, thereby organizing tissues and participating in their homeostasis. There are many pathways leading to cell death that are activated by numerous external and/or internal stimuli.¹ Cell death can occur via programed processes such as apoptosis, autophagy, necrosis (a more passive process) or necroptosis (a type of programed necrosis).²During apoptosis, specific gene networks and protein-cleavage programs are activated sending the cells on a death spiral^{3, 4, 5} through a family of cysteine-aspartate proteases (caspases).⁶ Caspases are classified by their role in the apoptotic pathway, into (i) initiator caspases (such as caspases2, 8, 9 and 10) or (ii) effector caspases (such as caspases3, 6 or 7).⁷ Initiator caspases cleave the inactive precursor of the effector caspases (pro-caspases) into their active forms. Effector caspases are then responsible for cleaving protein targets to interfere with cellular processes, and in particular, with the activation of some endonucleases that degrade genomic DNA.Throughout the process of mRNA maturation, numerous quality control mechanisms verify the integrity of the information carried by mRNAs. One of these, nonsense-mediated mRNA decay (NMD), leads to the rapid decay of mRNAs harboring a premature termination codon (PTC) to prevent the synthesis of non-functional and/or potentially detrimental truncated proteins.^{8, 9, 10} In addition to its role in quality control, NMD also regulates gene expression of so-called natural substrates of NMD.^{11, 12, 13, 14, 15} Proteins that have a central role in NMD, such as UPF1, UPF2, UPF3/UPF3a and UPF3X/UPF3b are highly conserved from yeast to human. The requirement for these UPF proteins in NMD is illustrated by the fact that the downregulation of any one of them results in an inhibition of NMD.^{16, 17} NMD is a necessary component in the development and maintenance of healthy cells and organisms. For example, UPF1 is an essential gene since the inactivation of UPF1 protein leads to an early embryonic death in mouse,¹⁸ thus implicating NMD as a critical proofreading and/or regulatory component in early organismal development. However, the question of whether NMD is required after cells have committed to proceed along a pathway that culminates in cell death has not been investigated. To address this question, NMD efficiency was studied during apoptosis. The studies presented here show that NMD factors UPF1 and UPF2 are cleaved by caspases 3 and 7 during apoptosis. The functional consequences of these cleavages are a general shutdown of NMD activity leading to stabilization of both PTC-containing mRNAs and natural substrates of NMD, and also the production of caspase-cleaved UPF fragments that induce apoptosis and inhibit NMD.     

Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3'UTRs, whereas a distinct pathway degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2 and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. The molecular mechanism of long 3'UTR-based plant NMD resembled yeast NMD, whereas the intron-based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG-7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms.     

Assessing the activity of nonsense-mediated mRNA decay in lung cancer

Background

Inhibition of nonsense-mediated mRNA decay (NMD) in tumor cells can suppress tumor growth through expressing new antigens whose mRNAs otherwise are degraded by NMD. Thus NMD inhibition is a promising approach for developing cancer therapies. Apparently, the success of this approach relies on the basal NMD activity in cancer cells. If NMD is already strongly inhibited in tumors, the approach would not work. Therefore, it is crucial to assess NMD activity in cancers to forecast the efficacy of NMD-inhibition based therapy.

Methods

Here we develop three metrics using RNA-seq data to measure NMD activity, and apply them to a dataset consisting of 72 lung cancer (adenocarcinoma) patients.

Results

We show that these metrics have good correlations, and that the NMD activities in adenocarcinoma samples vary among patients: some cancerous samples show significantly stronger NMD activities than the normal tissues while some others show the opposite pattern. The variation of NMD activities among these samples may be partly explained by the varying expression of NMD effectors.

Conclusions

In sum, NMD activity varies among lung cancerous samples, which forecasts varying efficacies of NMD-inhibition based therapy. The developed metrics can be further used in other cancer types to assess NMD activity.

     

The role of SMG-1 in nonsense-mediated mRNA decay

SMG-1, a member of the PIKK (phosphoinositide 3-kinase related kinases) family, plays a critical role in the mRNA quality control system termed nonsense-mediated mRNA decay (NMD). NMD protects the cells from the accumulation of aberrant mRNAs with premature termination codons (PTCs) that encode nonfunctional or potentially harmful truncated proteins. SMG-1 directly phosphorylates Upf1, another key component of NMD, and this phosphorylation occurs upon recognition of PTC on post-spliced mRNA during the initial round of translation. At present, a variety of tools are available that can specifically suppress NMD, and it is possible to examine the contribution of NMD in a variety of physiological and pathological conditions.