Arabidopsis UPF1 RNA helicase for nonsense-mediated mRNA decay is involved in seed size control and is essential for growth

UPF1 RNA helicase plays a central role in nonsense-mediated mRNA decay (NMD), which specifically recognizes aberrant mRNAs containing premature termination codons and targets them for degradation. Although NMD factors are highly conserved among eukaryotes, little is known about the role of NMD in plant growth and development. The lba1 mutant of Arabidopsis thaliana with a Gly(851)-->Glu missense mutation in AtUPF1 yielded seeds that were on average 22% longer in the long axis and 35% heavier than the wild-type Col seeds. Expression of the wild-type AtUPF1 in this mutant reduced the seeds to a normal size. During early stages of seed development, globular to torpedo stages of the embryos were contained within seed sacs that were larger in lba1 than in Col. Furthermore, the distance between seeds in siliques was greater in lba1 than in Col, suggesting that the lba1 mutation may affect ovule development. Self-pollinated atupf1-3(+/-) plants heterozygous for AtUPF1 disrupted by T-DNA insertion developed atupf1-3(-/-) seeds with a size and shape similar to those of Col seeds. However, the atupf1-3(-/-) seedlings stopped growing after radicle emergence from the seed coat, and this seedling lethality was rescued by expressing the wild-type AtUPF1. These results suggest that the lba1 mutation in AtUPF1 maternally affects seed development and that AtUPF1 is essential for seedling growth.     

UPF3 suppresses aberrant spliced mRNA in Arabidopsis

It has been reported that eukaryotic organisms have a nonsense-mediated mRNA decay (NMD) system to exclude aberrant mRNAs that produce truncated proteins. NMD is an RNA surveillance pathway that degrades mRNAs possessing premature translation termination codons (PTCs), thus avoiding production of possibly toxic truncated proteins. Three interacting proteins, UPF1, UPF2 and UPF3, are required for NMD in mammals and yeasts, and their amino acid sequences are well conserved among most eukaryotes, including plants. In this study, 'The Arabidopsis Information Resource' database was searched for mRNAs with premature termination codons. We selected five of these mRNAs and checked for the presence of PTCs in these mRNAs when translated in vivo. As a result we identified aberrant mRNAs produced by alternative splicing for each gene. These genes produced at least one alternative splicing variant including a PTC (PTC+) and another variant without a PTC (PTC-). We analyzed their PTC+/PTC- ratios in wild-type Arabidopsis and upf3 mutant plants and showed that the PTC+/PTC- ratios were higher in atupf3 mutant plants than wild-type plants and that the atupf3 mutant was less able to degrade mRNAs with premature termination codons than wild-type plants. This indicated that the AtUPF3 gene is required by the plant NMD system to obviate aberrantly spliced mRNA.     

Interactions between UPF1, eRFs, PABP and the exon junction complex suggest an integrated model for mammalian NMD pathways

Nonsense-mediated mRNA decay (NMD) represents a key mechanism to control the expression of wild-type and aberrant mRNAs. Phosphorylation of the protein UPF1 in the context of translation termination contributes to committing mRNAs to NMD. We report that translation termination is inhibited by UPF1 and stimulated by cytoplasmic poly(A)-binding protein (PABPC1). UPF1 binds to eRF1 and to the GTPase domain of eRF3 both in its GTP- and GDP-bound states. Importantly, mutation studies show that UPF1 can interact with the exon junction complex (EJC) alternatively through either UPF2 or UPF3b to become phosphorylated and to activate NMD. On this basis, we discuss an integrated model where UPF1 halts translation termination and is phosphorylated by SMG1 if the termination-promoting interaction of PABPC1 with eRF3 cannot readily occur. The EJC, with UPF2 or UPF3b as a cofactor, interferes with physiological termination through UPF1. This model integrates previously competing models of NMD and suggests a mechanistic basis for alternative NMD pathways.     

Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2x10(-16)). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.     

The cap-snatching frequency of a plant bunyavirus from nonsense mRNAs is low but is increased by silencing of UPF1 or SMG7

Bunyaviruses cleave host cellular mRNAs to acquire cap structures for their own mRNAs in a process called cap-snatching. How bunyaviruses interact with cellular mRNA surveillance pathways such as nonsense-mediated decay (NMD) during cap-snatching remains poorly understood, especially in plants. Rice stripe virus (RSV) is a plant bunyavirus threatening rice production in East Asia. Here, with a newly developed system allowing us to present defined mRNAs to RSV in Nicotiana benthamiana, we found that the frequency of RSV to target nonsense mRNAs (nsRNAs) during cap-snatching was much lower than its frequency to target normal mRNAs. The frequency of RSV to target nsRNAs was increased by virus-induced gene silencing of UPF1 or SMG7, each encoding a protein component involved in early steps of NMD (in an rdr6 RNAi background). Coincidently, RSV accumulation was increased in the UPF1- or SMG7-silenced plants. These data indicated that the frequency of RSV to target nsRNAs during cap-snatching is restricted by NMD. By restricting the frequency of RSV to target nsRNAs, NMD may impose a constraint to the overall cap-snatching efficiency of RSV. Besides a deeper understanding for the cap-snatching of RSV, these findings point to a novel role of NMD in plant–bunyavirus interactions.