The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens.
ABSTRACT: Nonsense-mediated mRNA decay (NMD) is important for RNA quality control and gene regulation in eukaryotes. NMD targets aberrant transcripts for decay and also directly influences the abundance of non-aberrant transcripts. In animals, the SMG1 kinase plays an essential role in NMD by phosphorylating the core NMD factor UPF1. Despite SMG1 being ubiquitous throughout the plant kingdom, little is known about its function, probably because SMG1 is atypically absent from the genome of the model plant, Arabidopsis thaliana. By combining our previously established SMG1 knockout in moss with transcriptome-wide analysis, we reveal the range of processes involving SMG1 in plants. Machine learning assisted analysis suggests that 32% of multi-isoform genes produce NMD-targeted transcripts and that splice junctions downstream of a stop codon act as the major determinant of NMD targeting. Furthermore, we suggest that SMG1 is involved in other quality control pathways, affecting DNA repair and the unfolded protein response, in addition to its role in mRNA quality control. Consistent with this, smg1 plants have increased susceptibility to DNA damage, but increased tolerance to unfolded protein inducing agents. The potential involvement of SMG1 in RNA, DNA and protein quality control has major implications for the study of these processes in plants.
Project description:Nonsense-mediated decay (NMD) is a messenger RNA quality-control pathway triggered by SMG1-mediated phosphorylation of the NMD factor UPF1. In recent times, the RNA helicase DHX34 was found to promote mRNP remodelling, leading to activation of NMD. Here we demonstrate the mechanism by which DHX34 functions in concert with SMG1. DHX34 comprises two distinct structural units, a core that binds UPF1 and a protruding carboxy-terminal domain (CTD) that binds the SMG1 kinase, as shown using truncated forms of DHX34 and electron microscopy of the SMG1-DHX34 complex. Truncation of the DHX34 CTD does not affect binding to UPF1; however, it compromises DHX34 binding to SMG1 to affect UPF1 phosphorylation and hence abrogate NMD. Altogether, these data suggest the existence of a complex comprising SMG1, UPF1 and DHX34, with DHX34 functioning as a scaffold for UPF1 and SMG1. This complex promotes UPF1 phosphorylation leading to functional NMD.
Project description:Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.
Project description:PI3K-related kinases (PIKKs) are large Serine/Threonine (Ser/Thr)-protein kinases central to the regulation of many fundamental cellular processes. PIKK family member SMG1 orchestrates progression of an RNA quality control pathway, termed nonsense-mediated mRNA decay (NMD), by phosphorylating the NMD factor UPF1. Phosphorylation of UPF1 occurs in its unstructured N- and C-terminal regions at Serine/Threonine-Glutamine (SQ) motifs. How SMG1 and other PIKKs specifically recognize SQ motifs has remained unclear. Here, we present a cryo-electron microscopy (cryo-EM) reconstruction of a human SMG1-8-9 kinase complex bound to a UPF1 phosphorylation site at an overall resolution of 2.9 Å. This structure provides the first snapshot of a human PIKK with a substrate-bound active site. Together with biochemical assays, it rationalizes how SMG1 and perhaps other PIKKs specifically phosphorylate Ser/Thr-containing motifs with a glutamine residue at position +1 and a hydrophobic residue at position -1, thus elucidating the molecular basis for phosphorylation site recognition.
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved mRNA quality control mechanism that identifies and destroys aberrant mRNAs that contain premature termination codons (PTCs). The NMD core comprises seven proteins, from SMG1 to SMG7. Arabidopsis has orthologues of most of these proteins. Studies on yeast, Drosophila, Humans and Arabidopsis reveal that NMD not only functions to target PTC-containing mRNAs but also acts as a global regulator of gene expression. It has also been reported that the NMD pathway branches with some target genes being dependent on specific NMD factors. In order to determine the extent to which different NMD factors co-regulate or independently regulate Arabidopsis genes, transcriptome analysis of smg7b-1 mutants will be carried out and added to existent data of upf1, upf3 and smg5 NMD mutants for comparison. RNA will be extracted from 17 day-old mutant and wild type seedlings grown at 22-24 C under constant light. 4 samples were used in this experiment
Project description:Translation-dependent mRNA quality control systems protect the protein homeostasis of eukaryotic cells by eliminating aberrant transcripts and stimulating the decay of their protein products. Although these systems are intensively studied in animals, little is known about the translation-dependent quality control systems in plants. Here, we characterize the mechanism of nonstop decay (NSD) system in Nicotiana benthamiana model plant. We show that plant NSD efficiently degrades nonstop mRNAs, which can be generated by premature polyadenylation, and stop codon-less transcripts, which are produced by endonucleolytic cleavage. We demonstrate that in plants, like in animals, Pelota, Hbs1 and SKI2 proteins are required for NSD, supporting that NSD is an ancient and conserved eukaryotic quality control system. Relevantly, we found that NSD and RNA silencing systems cooperate in plants. Plant silencing predominantly represses target mRNAs through endonucleolytic cleavage in the coding region. Here we show that NSD is required for the elimination of 5' cleavage product of mi- or siRNA-guided silencing complex when the cleavage occurs in the coding region. We also show that NSD and nonsense-mediated decay (NMD) quality control systems operate independently in plants.
Project description:SMG1 is a member of the phosphoinositide kinase-like kinase family of proteins that includes ATM, ATR, and DNA-PK, proteins with known roles in DNA damage and cellular stress responses. SMG1 has a well-characterized role in nonsense-mediated decay as well as suggested roles in the DNA damage response, resistance to oxidative stress, regulation of hypoxic responses, and apoptosis. To understand the roles of SMG1 further, we generated a Genetrap Smg1 mouse model. Smg1 homozygous KO mice were early embryonic lethal, but Smg1 heterozygous mice showed a predisposition to a range of cancers, particularly lung and hematopoietic malignancies, as well as development of chronic inflammation. These mice did not display deficiencies in known roles of SMG1, including nonsense-mediated decay. However, they showed elevated basal tissue and serum cytokine levels, indicating low-level inflammation before the development of tumors. Smg1 heterozygous mice also showed evidence of oxidative damage in tissues. These data suggest that the inflammation observed in Smg1 haploinsufficiency contributes to susceptibility to cancer and that Smg1-deficient animals represent a model of inflammation-enhanced cancer development.
Project description:Nonsense-mediated decay (NMD) is a eukaryotic quality control pathway, involving conserved proteins UPF1, UPF2 and UPF3b, which detects and degrades mRNAs with premature stop codons. Human UPF2 comprises three tandem MIF4G domains and a C-terminal UPF1 binding region. MIF4G-3 binds UPF3b, but the specific functions of MIF4G-1 and MIF4G-2 are unknown. Crystal structures show that both MIF4G-1 and MIF4G-2 contain N-terminal capping helices essential for stabilization of the 10-helix MIF4G core and that MIF4G-2 interacts with MIF4G-3, forming a rigid assembly. The UPF2/UPF3b/SMG1 complex is thought to activate the kinase SMG1 to phosphorylate UPF1 in vivo. We identify MIF4G-3 as the binding site and in vitro substrate of SMG1 kinase and show that a ternary UPF2 MIF4G-3/UPF3b/SMG1 complex can form in vitro. Whereas in vivo complementation assays show that MIF4G-1 and MIF4G-2 are essential for NMD, tethering assays reveal that UPF2 truncated to only MIF4G-3 and the UPF1-binding region can still partially accomplish NMD. Thus UPF2 MIF4G-1 and MIF4G-2 appear to have a crucial scaffolding role, while MIF4G-3 is the key module required for triggering NMD.
Project description:Nonsense-mediated-decay (NMD) is a eukaryotic RNA surveillance mechanism that controls the levels of both aberrant and normal transcripts. The regulation of this process is not well understood. The Arabidopsis NMD factor UPF3 is regulated by a negative feedback-loop that targets its own transcript for NMD. We investigated the functional significance of this control for the overall regulation of NMD in Arabidopsis. For this, we tested the ability of NMD-sensitive and -insensitive forms of UPF3, expressed under the control of UPF3 promoter, to complement NMD functionality in NMD-mutant plants and investigated their impact in wild-type (WT) plants. The sensitivity of UPF3 transcript to NMD was essential for efficient complementation of NMD in upf3 mutants. Upregulated UPF3 expression in WT plants resulted in over-degradation of certain transcripts and inhibited degradation of other transcripts. Our results demonstrate that, in contrast to mammalian cells, a delicate balance of UPF3 transcript levels by its feedback loop and by restriction of its transcription, are crucial for proper NMD regulation in Arabidopsis. Interestingly, the levels of many small-nucleolar-RNAs (snoRNAs) were decreased in upf1 and upf3 mutants and increased upon enhanced UPF3 expression. This suggests that proper snoRNA homeostasis in Arabidopsis depends on the integrity of the NMD pathway.
Project description:Nonsense-mediated decay (NMD) is a surveillance mechanism that degrades aberrant mRNAs. A complex comprising SMG1, UPF1, and the translation termination factors eRF1 and eRF3 (SURF) is assembled in the vicinity of a premature termination codon. Subsequently, an interaction with UPF2, UPF3b, and the exon junction complex induces the formation of the decay-inducing complex (DECID) and triggers NMD. We previously identified the RNA helicase DHX34 as an NMD factor in C. elegans and in vertebrates. Here, we investigate the mechanism by which DHX34 activates NMD in human cells. We show that DHX34 is recruited to the SURF complex via its preferential interaction with hypophosphorylated UPF1. A series of molecular transitions induced by DHX34 include enhanced recruitment of UPF2, increased UPF1 phosphorylation, and dissociation of eRF3 from UPF1. Thus, DHX34 promotes mRNP remodeling and triggers the conversion from the SURF complex to the DECID complex resulting in NMD activation.
Project description:Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades mRNAs containing nonsense codons, and regulates the expression of naturally occurring transcripts. While NMD is not essential in yeast or nematodes, UPF1, a key NMD effector, is essential in mice. Here we show that NMD components are required for cell proliferation in Drosophila. This raises the question of whether NMD effectors diverged functionally during evolution. To address this question, we examined expression profiles in Drosophila cells depleted of all known metazoan NMD components. We show that UPF1, UPF2, UPF3, SMG1, SMG5, and SMG6 regulate in concert the expression of a cohort of genes with functions in a wide range of cellular activities, including cell cycle progression. Only a few transcripts were regulated exclusively by individual factors, suggesting that these proteins act mainly in the NMD pathway and their role in mRNA decay has not diverged substantially. Finally, the vast majority of NMD targets in Drosophila are not orthologs of targets previously identified in yeast or human cells. Thus phenotypic differences observed across species following inhibition of NMD can be largely attributed to changes in the repertoire of regulated genes.