Unique Aspects of Plant Nonsense-Mediated mRNA Decay

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Review

Unique Aspects of Plant Nonsense-mediated mRNA Decay Orit Shaul* Nonsense-mediated mRNA Decay (NMD) is a eukaryotic quality-control mechanism that governs the stability of both aberrant and normal transcripts. Although plant and mammalian NMD share great similarity, they differ in certain mechanistic and regulatory aspects. Whereas SMG6 (from Caenorhabditis elegans ‘suppressor with morphogenetic effect on genitalia’)-catalyzed endonucleolytic cleavage is a prominent step in mammalian NMD, plant NMD targets are degraded by an SMG7-induced exonucleolytic pathway. Both mammalian and plant NMD are downregulated by stress, thereby enhancing the expression of defense response genes. However, the target genes and processes affected differ. Several plant and mammalian NMD factors are regulated by negative feedback-loops. However, while the loop regulating UPF3 (up-frameshift 3) expression in not vital for mammalian NMD, the sensitivity of UPF3 to NMD is crucial for the overall regulation of plant NMD.

Trends NMD shapes the plant transcriptome. Plant and mammalian NMD share similarities, but differ in mechanistic and regulatory aspects. NMD inhibition during pathogen attack elicits plant defense responses. Plant NMD is controlled by feedback regulation of its key factors.

NMD Determines the Fate of Many Normal and Aberrant Plant Transcripts Gene expression is determined, to a large extent, by post-transcriptional events. NMD is one of several quality-control mechanisms that couple aberrant translation elongation or termination to accelerated RNA decay (reviewed in [1–3]). NMD eliminates aberrant transcripts that contain premature termination codons (PTCs), thereby preventing the accumulation of truncated, potentially deleterious proteins (reviewed in [4–9]). The NMD process has a profound impact on the plant transcriptome [10–12]. It degrades transcripts derived from pseudogenes, transposable elements, potential natural antisense RNAs, and aberrant mRNA-like non-coding RNAs [12]. Moreover, NMD controls the levels of many normal protein-coding mRNAs. These mRNAs include alternatively spliced transcript isoforms [10,11] and other normal transcripts with cis elements that may lead to the recognition of their termination codons (TCs) as premature. Owing to the large number of transcripts that can be affected by NMD, understanding how this process is executed and regulated can shed more light on the control of gene expression and plant physiology. The basic features of NMD are conserved among eukaryotes, and have been the subject of several recent reviews [4–9]; they are therefore not described here in detail. Following a brief summary of the current knowledge about NMD in mammalian cells and the common features in plant cells, this review will mainly focus on aspects that are unique to plant NMD. Orthologs (see Glossary) of several mammalian NMD factors are also functional in plants, whereas other mammalian NMD factors, particularly those directly involved in target RNA degradation, have not yet been identified in plants. NMD is downregulated in response to stress in both mammals and plants, but the target genes and the affected processes differ. The contribution of NMD inhibition during biotic stress to the development of immunity responses is described. While

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The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel

*Correspondence: [email protected] (O. Shaul).

http://dx.doi.org/10.1016/j.tplants.2015.08.011 © 2015 Elsevier Ltd. All rights reserved.

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several plant and mammalian NMD factors are controlled by negative feedback loops, there is a difference in the significance of UPF3 regulation in the two groups of organisms. This review highlights that, despite their basic similarity, plant and mammalian NMD differ in some mechanistic and regulatory aspects.

Mammalian NMD: Current Models NMD uses a set of factors to sense the integrity of translation termination and eliminate transcripts with PTCs or features that lead to the recognition of TCs as premature (reviewed in [4–8]). The NMD factors UPF1, UPF2, and UPF3 are conserved in all eukaryotes. Multicellular eukaryotes have additional NMD factors, including SMG proteins. The NMD mechanism in mammalian cells has been extensively studied (reviewed in [5,7–9]). Figure 1 illustrates mammalian NMD occurring at the steady-state rounds of translation (see [5] for a model of mammalian NMD during the pioneer round of translation). The cap structure (7-methyl guanosine, 7mG) of the mRNA interacts during the pioneer and standard rounds of translation with protein complexes termed the cap-binding complex (CBC) and the eukaryotic initiation factor 4F (eIF4F), respectively. In the eIF4F complex, the cap-binding subunit eIF4E binds to eIF4G. During the steady-state rounds of translation, interaction between eIF4G and the poly (A)-binding protein cytoplasmic 1 (PABPC1) leads to a circular conformation of the mRNA (Figure 1A). Upon reaching a TC, the ribosome binds to the eukaryotic release factors eRF1 and eRF3. Normal translation termination and ribosome recycling, which prohibit NMD, depend on the interaction of eRF3 with the eIF4G-bound PABPC1 [13,14] (Figure 1A). When the 30 untranslated region (30 UTR) of the mRNA is long, eRF3–PABPC1 interaction is inhibited owing to the large distance between them (Figure 1B). This prevents efficient translation termination and ribosome recycling [13,14]. Consequently, eRF3 interacts with the ATP-dependent RNA helicase UPF1 to form the SURF complex, which also includes eRF1 and the protein kinase SMG1 [15] (Figure 1B). SMG1 interacts with the regulatory subunits SMG8 and SMG9, which inhibit its kinase activity before it is activated by the subsequent events [16]. The interaction between the SURF complex and the NMD factors UPF2 and UPF3 leads to the activation of SMG1, which phosphorylates UPF1 [15,17,18] (Figure 1C). The interaction of UPF1 with UPF2 also triggers the helicase activity of UPF1 [19]. Phosphorylated UPF1 triggers translational repression and recruits the endonuclease SMG6, which cleaves the RNA (reviewed in [7,20]) (Figure 1C). The 50 cleavage product is subjected to 30 -to-50 decay, possibly by the exosome. UPF1 helicase activity disassembles proteins bound to the 30 cleavage product, facilitating 50 -to30 exonucleolytic degradation by 50 –30 exoribonuclease 1 (XRN1). Phosphorylated UPF1 also recruits SMG5 that binds to either the SMG7 or nuclear receptor coregulatory protein 2 (PNRC2) proteins (Figure 1C), resulting in mRNA decapping, followed by 50 -to-30 degradation by XRN1 and/or deadenylation followed by 30 -to-50 degradation (reviewed in [7,20]). The SMG5–SMG7 complex recruits protein phosphatase 2A, which dephosphorylates and recycles UPF1. Introns located 50–55 nt downstream of TCs facilitate NMD [21,22]. Following splicing, a complex of proteins, the exon-junction complex (EJC), is deposited on the mRNA 24 nt upstream of exon–exon junctions (EEJs). UPF3, a nucleocytoplasmic shuttling protein, interacts with the EJC before export of the mRNA to the cytoplasm [23,24]. If termination occurs 50– 55 nt upstream of an EEJ, the size of the terminating ribosome is insufficient to physically remove the EJC. The EJC-bound UPF3 then interacts with UPF2, which bridges between UPF3 and UPF1 [25] (Figure 1B), thereby facilitating UPF1 recruitment to the mRNA and NMD activation. UPF1 can also bind to the mRNA before translation in a 30 UTR length-dependent manner [26], and be displaced to the 30 UTR by the translating ribosome [27–29] and/or translocate along the transcript by utilizing its RNA helicase activity [30]. The fact that UPF1 is enriched in long 30 UTRs [26–29] explains why, although introns 50–55 nt downstream of TCs increase NMD efficiency, they are not essential for it, and NMD can be activated by long 30 UTRs alone. NMD can also be

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Glossary Cap-binding complex (CBC): consists of 20 and 80 kDa polypeptides termed CBP20 (capbinding protein 20) and CBP80, respectively. Deadenylation: shortening of the 30 poly(A) tail. Decapping: hydrolysis of the mRNA cap structure. Exosome: a 30 –50 exoribonuclease complex involved in RNA processing and degradation. Helicase activity: unwinding of double-stranded DNA or RNA. Hypomorphic mutation: a mutation that causes a partial loss of gene function, either through reduced RNA or protein expression, or through reduced functional performance. Hypoxia: diminished availability of oxygen to the body tissues. Kozak context: in eukaryotes, the efficiency of initiation-codon recognition by the scanning ribosome is dependent on the so-called Kozak context. The most crucial sites with respect to the start codon (where the A in the AUG is +1) are 3 and +4, usually occupied by a purine and guanine, respectively. 7-Methylguanosine (7mG): the cap structure into which the first transcribed nucleotide of RNA polymerase II transcripts is modified during the early stages of transcription. Nuclear receptor coregulatory protein 2 (PNRC2): a bifunctional protein that both regulates transcription and controls NMD (by promoting decapping). Null mutation: a mutation that results in the complete loss of function for a particular gene. Orthologs: genes in different species that evolved from a common ancestral gene and usually retain the same function. Poly(A)-binding protein cytoplasmic 1 (PABPC1): a mammalian protein that binds to the poly(A) tail of the mRNA in the cytoplasm, and replaces the nuclear poly(A)-binding protein PABPN1 following mRNA export to the cytosol. Pioneer round of translation: the first round of translation of the mRNA after its export to the cytoplasm. This is accompanied by remodeling of the messenger ribonucleoprotein complex (mRNP) in processes that either are or are not augmented by

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Figure 1. Model of Mammalian Nonsense-mediated mRNA Decay (NMD). The model illustrates NMD occur-

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ring during the steady-state rounds of translation, and is based on the studies and models cited in the text. NMD factors that have not yet been identified in plants (SMG5, SMG6, SMG8, SMG9, and PNRC2) are presented as grey shapes. (A) Normal translation termination and ribosome recycling, which prohibit NMD. The cap structure (7mG) of the mRNA is bound by eIF4E, which binds to eIF4G. The interaction between eIF4G and PABPC1, which is bound to the poly(A) tail of the mRNA, leads to a circular conformation of the mRNA. Proximity of the termination codon (TC) to the poly(A) tail allows efficient interaction between eRF3, which binds to the terminating ribosome, and PABPC1–eIF4G. This allows efficient translation termination and ribosome recycling, which prevent NMD. (B) Aberrant translation termination and the early steps of NMD. A long distance between the TC and the poly(A) tail prevents complex formation between eRF3 and PABPC1– eIF4G. This prevents normal translation termination and ribosome recycling, and facilitates the formation of the SURF complex including eRF1–eRF3, UPF1, and SMG1 (SMG1 is bound by the inhibitory subunits SMG8 and SMG9). UPF2 bridges between UPF1 and free or EJC-bound UPF3. Because UPF3 is present in EJCs, the endurance of an EJC downstream of a terminating ribosome facilitates the recruitment of UPF2–UPF1–SMG1, and leads to NMD even when the 30 UTR is not exceptionally long. The EJC is indicated by a dashed ellipse because it is not essential for NMD, which can be activated by long 30 UTRs alone (see text). (C) The late steps of NMD. The interaction between the SURF complex and UPF2–UPF3 leads to activation of SMG1 (accompanied by SMG8– SMG9 release), which phosphorylates UPF1. Phosphorylated UPF1 triggers translational repression, and binds SMG6 and SMG5 on phosphorylated sites (indicated by ‘P’) located at its N- and C-termini, respectively. This is accompanied by the dissociation of the ribosome and release factors. SMG6 endonucleolytically cleaves the mRNA, while complexes of SMG5 with either SMG7 or PNRC2 recruit decapping and/or deadenylating enzymes. See text for more details.

translation, including CBC replacement by eIF4E, PABPN1 replacement by PABPC1, and EJC removal [5]. Reinitiation: the resumption of translation following termination. Steady-state rounds of translation: the rounds of translation that follow the pioneer round of translation and associated mRNP remodeling. Tethering: an approach for studying the consequences of artificially binding (tethering) a protein to an mRNA. This is achieved by the coexpression of two recombinant molecules: first, an mRNA including a sequence element recognized by a well-characterized RNA-binding protein, and second, an mRNA encoding the protein to be bound fused to the RNA-binding domain of the indicated RNA-binding protein.

activated by upstream open reading frames (uORFs), whose termination codons can be recognized as premature owing to their long distance from the poly(A) tail. However, not every gene with features that can potentially trigger NMD is subject to NMD [31], and NMD-inducing features are often undefined [32]. Genes upregulated in mutants of NMD factors can be either

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direct NMD targets or indirect targets of this process, whose expression is affected by the changes in direct targets.

Similarities and Differences Between Plant and Mammalian NMD The basic features of mammalian NMD are conserved in plants (reviewed in [3,33,34]). Orthologs of mammalian UPF1, UPF2, UPF3, SMG1, SMG7, and EJC proteins are also functional in plant NMD [35–45]. While SMG1 is present in most plants, it is absent in Arabidopsis [38]. It was deduced that Arabidopsis UPF1 is phosphorylated by an alternative kinase [38]. Similarly to mammals, plant transcripts are targeted to NMD by PTCs [35,36,41], long (300–350 nt) 30 UTRs [11,46–48], introns 50–55 nt downstream of TCs [39,46,47], and uORFs [11,49–51]. The extent to which uORFs expose their transcripts to NMD is not yet clear. Several features enhance the likelihood that an uORF-containing plant gene will be targeted to NMD. The first feature is a long (>35–50 aa) uORF peptide [49]. Similar observations in mammalian cells were explained by models in which, after translating a short uORF, the terminating ribosome remains close to PABPC1 owing to mRNA circularization, and, in addition, reinitiation is more efficient [52]. It was shown that reinitiation can prevent NMD [53]. The second feature that was associated with NMD of uORF-containing plant genes is uORF overlap with the main start codon [11], probably owing to a lower probability of reinitiation. The third feature is an uORF with a conserved peptide (CPuORF) [51]. The conservation of CPuORFs peptides suggests that they are translated and, hence, may lead to NMD [51]. NMD is also triggered in rare cases in which the uORF peptide stalls the ribosome at its TC, as seen for example in the Arabidopsis AdoMetDC1 gene [54], and as previously identified in yeast (Saccharomyces cerevisiae) [55] and other eukaryotes. Although long uORFs are more likely to elicit NMD, even a short uORF of only 13 aa mediated efficient NMD of the Arabidopsis MHX gene [50], possibly because the efficiency of reinitiation following its translation was low [56]. Moreover, this uORF was efficiently translated despite the weak Kozak context of its upstream AUG (uAUG) codon [56,57]. The fact that even a short uORF with a weak Kozak-context uAUG can efficiently target its transcript for NMD demonstrates that the features that expose uORFcontaining transcripts to NMD are not always apparent. Several studies have suggested that, despite their basic similarity, there are also differences in the way NMD is executed in plant and mammalian cells. Orthologs of several mammalian NMD factors, including SMG5, SMG6, SMG8, SMG9, and PNRC2, have not been identified thus far in plants [45] (these factors are presented as grey shapes in Figure 1). Whereas SMG6-catalyzed endonucleolytic cleavage is a prominent step in human NMD [58,59], plant NMD targets are apparently degraded by an SMG7-induced exonucleolytic pathway [60]. In mammalian cells, the simultaneous binding of SMG6 and the SMG5–SMG7 complex to phosphorylated sites at the N- and C-terminal regions, respectively, of UPF1 (Figure 1C) is essential for both NMD and UPF1 dissociation from the mRNA [61]. While this simultaneous binding to both the N- and C-terminal regions of UPF1 is essential for mammalian NMD, the phosphorylated N- and C-terminal sites of plant UPF1 play redundant (but essential) roles in plant NMD [60]. It was suggested that the phosphorylated N- and C-terminal regions of plant UPF1 recruit SMG7 to the functional NMD complex, followed by target degradation [60]. In mammals, the exonuclease XRN1 is important for target degradation (see above). However, it was suggested that the plant homolog of XRN1, XRN4, is not essential for plant NMD [60]. Other differences between plant and mammalian NMD are apparently related to the function or necessity of commonly present factors. While EJC proteins are evidently involved in plant NMD [39,45], it was found that, unlike in mammals, the 30 UTR tethering of EJC proteins did not result in mRNA decay in plants [46]. In addition, most studies on mammalian cells indicate that NMD occurs rapidly after nuclear export, irrespective of whether the mRNA is bound by the CBC and

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undergoes the pioneer round of translation, or if it is bound by eIF4E (which replaces the CBC after the first round of translation) and undergoes steady-state rounds of translation (reviewed in [62]). During the pioneer round of translation of mammalian cells, UPF1 is directed to eRF3, which binds to the terminating ribosome, by the cap binding protein 80 (CBP80) subunit of CBC [63]. Silencing the mammalian CBP80 protein inhibited mammalian NMD [64]. However, NMD was not inhibited in Arabidopsis plants mutated in either CBP80 or CBP20 [65]. This indicates that the CBC is not essential for plant NMD [65]. In addition to the ‘classical’ branch of mammalian NMD described above, three other branches (that may have overlapping substrate specificities) were shown to have lower or no dependence on UPF2, UPF3, or the EJC ([66], reviewed in [67]). Human UPF3 can interact with UPF2 and with EJC proteins [68,69]. A mechanistic explanation for a mammalian NMD branch that does not depend on UPF2 was based on few studies showing direct interaction between UPF1 and UPF3, independently of UPF2 [69–71]. However, plant UPF3 was able to bind UPF2, but failed to immunoprecipitate with UPF1 in the absence of UPF2 [45]. It is thus possible that plants do not have a UPF2-independent NMD branch. The fact that no null mutant of UPF2 has been identified thus far in plants [38,40] is consistent with this possibility. Alternatively, such a branch may be spatially or temporally restricted. Alternative splicing of many genes generates isoforms with PTCs that are potential NMD substrates. Plant transcripts with retained introns (a common alternative-splicing event in plants) are often insensitive to NMD owing to their retention in the nucleus or nucleolus [11,37,72]. There are some reports of nuclear retention of mammalian genes that have retained introns ([73,74] and references therein). However, transcripts with retained introns are efficiently transported to the cytosol of white blood cells, followed by NMD of the transcripts [75]. Further research is necessary to learn whether there are fundamental differences between plant and mammalian cells with respect to the fate of transcripts with retained introns. The experimental evidence detailed here highlights some mechanistic points in which plant and mammalian NMD may differ. The precise reactions that underlie these differences, as well as their functional and physiological significance, are yet to be uncovered.

Cellular Processes Affected by NMD The NMD pathway controls not only aberrant and non-coding RNAs, but also normal transcripts. As such, it can directly and indirectly affect many cellular processes. Indeed, mammalian NMD targets were shown to be involved in various processes, including stem cell development, genomic stability, amino acid homeostasis, cell cycle, splicing, brain development, and stress responses (reviewed in [7,76–78]). Similarly, NMD controls the transcript levels of many genes that play important roles in plant development (e.g., [11,54,79–81], see also [82,83]). The possibility that NMD provides a means to collectively modulate the expression of multiple genes involved in a given process or pathway is particularly intriguing, and there are several examples. Genes encoding splicing regulators, such as serine/arginine (SR) proteins, are among the most conserved NMD substrates in animals (reviewed in [7,76]). High levels of the affected splicing regulators increase the abundance of PTC-containing, alternatively spliced versions of their own transcripts, triggering their downregulation in a negative feedback loop. Coupling of alternative-splicing of SR genes with NMD was also demonstrated in Arabidopsis [84]. This suggests that the role of NMD in splicing regulation is conserved. Alternative splicing coupled to NMD plays an important role in plant adaptation to stress [11,81–84]. Various stresses were shown to increase the number of alternatively spliced transcripts in Arabidopsis (see two recent reviews [82,83] for further details).

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Perhaps the most familiar example of NMD impacting on an entire physiological process in plants is related to pathogen defense responses. This subject is discussed in detail in the next section.

NMD Inhibition During Pathogen Attack Elicits Plant Defense Responses Both mammalian and plant NMD are downregulated in response to stress, thereby enhancing the expression of genes that assist in defense responses. However, the target genes, the processes affected, and the elicited responses are different in plant and mammalian cells. Mammalian NMD is downmodulated in response to various stress conditions, including hypoxia, deprivation of amino acids or other nutrients, infection, and the generation of reactive oxygen species (reviewed in [7,76,78]). The mechanism of NMD inhibition under these conditions is unknown, but involves phosphorylation of the eukaryotic initiation factor 2/ (eIF2/). Several NMD target transcripts promote adaptation to the indicated stresses [7,76,78]. Several observations led researchers to investigate the involvement of NMD in plant defense responses. These observations were made in Arabidopsis plants with hypomorphic or loss-offunction mutations in UPF1, UPF3, or SMG7. It was noticed that the phenotype of NMD mutants was similar to that of other Arabidopsis mutants that had enhanced pathogen resistance owing to constitutive upregulation of defense responses, including high levels of the plant hormone salicylic acid (SA) [85,86]. It was also noticed that the common genes upregulated in several NMD mutants had a large over-representation of genes related to pathogen responses mediated by SA [51]. It was revealed that Arabidopsis NMD mutants have higher levels of SA, compared to wild type (WT) plants, even in the absence of pathogen infection [51,85,86]. Increased SA levels (compared to WT plants) were also found in NMD mutants and UPF2-silenced (irUPF2) plants after infection with the virulent bacteria Pseudomonas syringae pv. tomato DC3000 (P. syringae) [40,51,85]. The NMD mutants also showed, under both non-infected and infected conditions, upregulated expression of genes encoding defense factors related to SA, including TIR-NB-LRR receptors, EDS1, PAD4, ICS1, NPR1, WRKY transcription factors, and pathogenesis-related (PR) proteins [51,85–87]. Figure 2 illustrates a model based on the known function of these defense proteins (reviewed in [88,89]), and on the studies detailed below [40,51,85–87,90], for the involvement of NMD in response to biotic stress. Box 1 provides a brief wider overview of the cellular events that follow pathogen attack in plants (reviewed in [88,89]). This wider overview is necessary for understanding the role of the NMD-affected defense proteins in the induction of immunity responses. In accordance with their increased levels of SA and defense proteins, NMD mutant plants exhibited elevated resistance to P. syringae [51,85,86]. It was also found that NMD is inhibited in WT plants infected with P. syringae [85,87]. Based on these findings, it was concluded that NMD

Box 1. A Brief Description of the Plant Response to Pathogens When plants are attacked by pathogens, molecules termed pathogen-associated molecular patterns (PAMPs) are detected by plant receptors and elicit PAMP-triggered immunity (PTI) (reviewed in [88,89]). Adapted (virulent) pathogens deliver the so-called ‘effector’ proteins inside host cells to suppress PTI. In resistant hosts, ETI is initiated by disease resistance (R) genes. The most common group of R genes encode nucleotide-binding site, leucine-rich repeat (NB-LRR) proteins. The NB-LRR proteins are divided into two subclasses on the basis of the presence of either a coiled-coil motif (CC-NB-LRRs) or a Toll/interleukin-1 receptor domain (TIR-NB-LRRs) in their N-termini. The NB-LRR proteins are intracellular receptors that recognize the pathogen-secreted effectors and activate ETI responses. These responses are mediated by increased levels of the hormone SA, and elevated expression of transcription factors containing the WRKY domain and of PR proteins. The local responses activated often lead to the hypersensitive response (HR), which leads to host cell death and prevents pathogen spreading. The onset of local responses usually leads to systemic acquired resistance (SAR), which is partially dependent on SA. SAR acts both locally and systemically, and confers longer protection against a secondary infection by a broad range of pathogens ([88,89] and references therein).

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Figure 2. Nonsense-mediated mRNA Decay (NMD) Inhibition During Biotic Stress Contributes to the Development of Immunity Responses. The model illustrates only the stress-related factors whose levels were reported to be affected by NMD [40,51,85–87,90], and the current knowledge about their interrelations ([85] and references therein; reviewed in [88,89]). See Box 1 for terms and abbreviations not defined here and for the integration of the NMD-affected factors presented here within a wider perspective of plant response to pathogens. Numbers in round brackets refer to the corresponding step in Figure 2. Biotic stress inhibits NMD (1). NMD is a negative regulator of expression of cytoplasmic TIRNB-LRR receptors (2) owing to NMD features in their genes. Consequently, biotic stress upregulates the expression of TIRNB-LRR receptors. The TIR-NB-LRR receptors bind to pathogen-secreted effectors, and, consequently, enhance the expression of the proteins enhanced disease susceptibility 1 (EDS1) and phytoalexin deficient 4 (PAD4) (3). The EDS1– PAD4 complex is essential (4) for salicylic acid (SA) synthesis from chorismate, which is catalyzed by iso-chorismatesynthase 1 (ICS1) (5). In a positive feedback loop (6), SA enhances the transcription of EDS1 and PAD4. SA also enhances the transcription of Nonexpressor-of-PR-genes-1 (NPR1) (7). In addition, SA enhances (8) the dissociation of NPR1 from an oligomeric into a monomeric form (9). The NPR1 monomer translocates from the cytosol to the nucleus and stimulates the transcriptional activation of defense-related genes, including genes encoding WRKY transcription factors (WRKY TFs) (10) and pathogenesis-related (PR) proteins (11). NPR1 also downregulates ICS1 expression, thereby reducing SA biosynthesis in a negative feedback loop (12). The WRKY TFs elevate the transcription of genes encoding both ICS1 (13) and PR proteins (14). SA activates the expression of PR proteins not only through an NPR1-dependent pathway, but also through NPR1independent pathways (15). PR proteins activate effector-triggered immunity (ETI) responses (Box 1) (16). In addition, SA induces ETI responses through PR proteins- and NPR1-independent pathways (17). The ETI responses increase the resistance to biotic stress (18). Positive and negative regulatory actions are indicated by arrows and lines with bars, respectively.

inhibition contributes to plant defense. It was subsequently deliberated how NMD inhibition, owing to either mutated NMD factors or pathogen infection, leads to enhanced resistance. Many of the defense genes upregulated in NMD mutants are indirect targets of NMD [51,90]. The involved primary targets were looked for by examination of the transcripts upregulated in the double-mutant smg7 pad4 [87]. PAD4 is part of the intracellular EDS1–PAD4 complex that is essential for effectortriggered immunity (ETI) responses [89] (Figure 2 and Box 1). The mutation in PAD4 prevents the development of immunity responses. This allowed the identification of transcripts whose upregulation in smg7 mutants was presumably the trigger, rather than the consequence, of immunityresponse activation. It was found that transcripts encoding TIR-NB-LRR proteins were significantly upregulated in smg7 pad4 mutants [87]. About 50% of the upregulated TIR-NB-LRR genes carried putative NMD features and had increased half-lives in the smg7 pad4 mutants. This indicates that NMD inhibition in plants mutated in NMD factors, or in P. syringae-infected plants, upregulates TIRNB-LRR transcripts, thereby enhancing plant ETI responses [87] (Figure 2).

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The Lethality of Severe NMD Impairment in Plants Results From Hyperactivation of Defense Responses NMD-deficient mutants of Saccharomyces cerevisiae and Caenorhabditis elegans are viable. However, severe NMD impairment in mice, zebrafish, or Drosophila is lethal ([91–93], reviewed in [6,8,77,94]). The affected animals had defects in multiple developmental processes, and it is currently unknown whether animal death resulted from a single or a combination of impaired processes. Moreover, in addition to their role in NMD, some NMD factors play a role in other cellular processes, including DNA synthesis, cell cycle progression, and the maintenance of telomeres (reviewed in [95,96]). Disruption of such NMD-unrelated processes could also contribute to animal lethality. While the precise cause of lethality of NMD-impaired animals is not yet clear, there are several indications that the aberrant growth and lethality of Arabidopsis mutants with severe NMD impairment results from hyperactivation of the plant defense system [86,87,90]. As discussed, NMD impairment in plants results in constitutive activation of SA-related defense responses [51,85–87]. All vegetative growth defects of smg7 mutants were suppressed in the doublemutant smg7 pad4, in which SA-related defense responses were impaired as a result of PAD4 disruption [86,87] (Figure 2). A null mutation in UPF1 leads to seedling lethality [35,42]. Nevertheless, the homozygous progeny of the cross between a upf1 null mutant and the pad4 mutant were viable [86]. In addition, the thin leaf phenotype of mutants in which UPF1 mRNA levels were severely reduced (but not completely eliminated) was suppressed in a double upf1 sid2-1 mutant that had a mutated allele of ICS1 (which encodes the enzyme that synthesize SA) and impaired SA accumulation [90].

NMD Regulation Compared to the current knowledge about NMD mechanism, much less is known about how NMD is regulated. Even so, there are indications that mammalian NMD is subject to developmental and tissue-specific controls, and can dynamically alter gene expression not only under stress conditions but also during normal growth (reviewed in [67,76,97]). A main component of NMD regulation is mediated by feedback loops that subject the transcripts of NMD factors themselves to NMD. Cells with mutated NMD factors, or in which the levels of NMD factors were depleted by RNA interference (RNAi) (i.e., cells whose NMD was weakened), often had increased levels of other factors of this pathway. Drosophila cells depleted of certain NMD factors had increased transcript levels of SMG5 and SMG6 [98]. UPF1-depleted human HeLa cells had increased levels of UPF2, SMG1, SMG5, SMG6, and SMG7 transcripts, while UPF1 was upregulated in cells depleted of the other NMD factors [99–101]. SMG7 expression was upregulated in Nicotiana benthamiana leaves in which UPF genes had been silenced [45] and in Arabidopsis NMD mutants [45,51]. The levels of Arabidopsis UPF3 mRNA were increased in upf1 mutant plants [50]. This indicates that the Arabidopsis SMG7 and UPF3 genes are regulated by negative feedback loops (Figure 3A). There is conflicting evidence as to whether UPF3 is targeted for feedback regulation in human cells. In contrast to other eukaryotes, vertebrates have two UPF3 genes, UPF3A and UPF3B, among which UPF3B encodes the main active isoform [23,102]. Increased UPF3B transcript levels were observed in human HeLa cells in which UPF1 was depleted by RNAi [100], whereas such an increase was not reported in another study in UPF1-depleted HeLa cells [101], nor was it seen in UPF1depleted mouse neuronal stem cells [100]. The NMD factors whose expression increases upon NMD suppression include features that can directly expose them to NMD. All human NMD factors whose transcript levels were increased in NMD-defective cells had long 30 UTRs, and some of them also had uORFs [100,101]. The Arabidopsis SMG7 transcript is targeted to NMD by a long 30 UTR and two introns downstream

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UPF3 expression Figure 3. Current Knowledge About Nonsense-mediated mRNA Decay (NMD) Regulation in Arabidopsis. (A) Expression of the Arabidopsis SMG7 and UPF3 genes is regulated by negative feedback loops. The NMD factors SMG7 and UPF3 contribute to NMD functionality. Augmented NMD downregulates the expression of the genes coding these NMD factors, resulting in a return to basal conditions [39,45,50,51,103]. Positive and negative regulatory actions are indicated by arrows and lines with bars, respectively. (B) Potential NMD inducing features in the genes of Arabidopsis NMD factors. Each ‘u’ in the 50 untranslated region (UTR) represents an upstream AUG (uAUG) codon. The ‘?’ refers to the lack in The Arabidopsis Information Resource 10 (TAIR10) of the 50 UTR sequence of UPF1. The lengths of the 30 UTRs (nt) are indicated. Introns in the 30 UTR are illustrated by triangles, with numbers that indicate the positions of the EEJs relative to the termination codons (TCs). Elements with experimentally validated impact on NMD of their transcripts are highlighted in pink. Long (300–350 nt) 30 UTRs are present in the Arabidopsis UPF1, UPF3, and SMG7 genes, but not in UPF2 ([45,103] and TAIR10). The long 30 UTRs of SMG7 and UPF3 were shown to target their transcripts to NMD [39,45,103]. Introns 50–55 nt downstream of TCs can elicit NMD. The intron 158 nt downstream the TC of SMG7 elicits NMD, while the intron 23 nt downstream the TC cannot elicit NMD, but is essential for correct splicing of the intron at the 158 nt position [39]. The intron 19 nt downstream of the TC of UPF3 cannot elicit NMD, probably owing to its proximity to the TC [103]. The uAUG codon of UPF3 does not elicit NMD [103]. The impact of the three uAUG codons of SMG7 is not known. (C) A model for the correlation between UPF3 expression and NMD efficiency in Arabidopsis. The model is based on that presented in [103]. It was shown that a delicate control of UPF3 expression by its feedback loop and by restriction of its transcription is crucial for NMD homeostasis in Arabidopsis [103]. The optimal level of UPF3 expression (which presumably varies between different cells and physiological conditions) is illustrated by zone B in the model. Zone A represents a range where UPF3 expression is rate-limiting for NMD. It was shown that even a moderate increase in UPF3 expression (presented by zone C) above the optimal level can inhibit NMD of certain transcripts [103]. This indicates that stringent control of UPF3 expression plays an important role in NMD regulation in Arabidopsis.

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of the TC [39,45] (Figure 3B). The Arabidopsis UPF3 gene is an NMD target because of a long 30 UTR [50,103]. The plant ortholog of the EJC component Barentsz is also an NMD target owing to an intron downstream of the TC [39]. It is also possible that the Arabidopsis UPF1 gene is an NMD target because it has a long (478 nt) 30 UTR, but this must be tested experimentally. In contrast to human UPF2 [100,101], the Arabidopsis UPF2 gene is apparently not controlled by an NMD feedback loop, nor does it have any potential NMD-inducing feature [103] (Figure 3B). These data indicate that NMD is regulated by feedback loops in which augmented NMD downregulates its own factors, resulting in a return to basal conditions. The physiological significance of this regulation was demonstrated in several cases. Elevated expression of human SMG1, SMG5, or SMG6 (but not UPF1, UPF2, or UPF3B) enhanced NMD efficiency, indicating that these factors are rate-limiting for human NMD [100]. Prevention of SMG1 upregulation in UPF1-depleted HeLa cells further abrogated NMD, while a moderate overexpression of SMG1 inhibited HeLa cell growth [100]. In Arabidopsis, expression in WT plants of a de-regulated form of UPF3 that could not be targeted by NMD, under the control of UPF3 promoter, caused a moderate (twofold) increase in UPF3 transcript levels [103]. This resulted in enhanced NMD of certain transcripts, indicating that UPF3 is rate-limiting for NMD of these transcripts, and that its feedback loop is essential for preventing undue overdegradation of these transcripts [103] (Figure 3C). Interestingly, microarray and model gene analysis showed that the moderate increase in UPF3 expression resulted in inhibited NMD of other Arabidopsis transcripts, including bona fide targets of this pathway [103] (Figure 3C). This indicates that a delicate balance of UPF3 transcript levels by its feedback loop plays a crucial role in NMD balancing in plants, and is essential for the prevention of either NMD inhibition or overactivation. These findings also indicate that there are differences in the factors that play a major role in balancing NMD in plant and mammalian cells. In human HeLa cells, even a 15- to 20-fold increase in the transcript levels of UPF3B did not enhance or inhibit NMD [100]. Overall, these data indicate that the role of UPF3 regulation in balancing NMD efficiency is more central in plant than in animal NMD. Expression in Arabidopsis smg7 mutants of a deregulated SMG7 version that could not be targeted by NMD, under the control of an actin promoter, elevated SMG7 transcript levels by twofold compared to WT plants [87]. This resulted in increased NMD efficiency of several NMD targets compared to WT plants, suggesting that SMG7 might be a rate-limiting factor for NMD of some plant transcripts [87]. To conclude, the feedback regulation of NMD factors is important for balancing NMD efficiency in plant as well as mammalian cells. However, the factors that play the major role in the maintenance of NMD homeostasis may differ in the two groups of organisms.

Concluding Remarks The basic features of NMD are conserved in plant and mammalian cells. However, there are apparently some differences in the mechanistic details in which this pathway is executed in the two groups of organisms. It will be interesting to fully understand the basis of these apparent differences and to evaluate whether they have any adaptive value for plants. It is also necessary to reveal whether other so far uncharacterized factors participate in plant NMD. Genes upregulated in mutants of NMD factors can be either direct or indirect targets of this process. As mentioned, the presence of potential NMD-inducing features in transcripts is not sufficient to differentiate between direct and indirect targets, and NMD-inducing features are often undefined [32]. Moreover, not every potential NMD feature leads to target degradation, as detailed above for uORFs and further supported by the recent identification of elements in

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Outstanding Questions Which plant genes are direct targets of the NMD process? Do some plant genes with long 30 UTRs include elements that inhibit NMD, similar to the elements recently identified in human genes? What underlies NMD inhibition during biotic stress? Is plant NMD affected by environmental cues other than biotic stress? Is NMD efficiency developmentally regulated in plants? In addition to the currently characterized NMD factors, do other proteins play mechanistic or regulatory roles in plant NMD?

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human long 30 UTRs that inhibit NMD [31]. Such elements have not been identified thus far in plants. In addition, in human cells, EJCs are present in most, but not all, EEJs, and many mapped EJCs do not localize to the canonical positions 24 nt upstream of EEJs (reviewed in [104]). This obstructs the reliable prediction of NMD-targeted transcripts on the basis of EEJ position information. Direct NMD targets were determined by several strategies in yeast, Drosophila, and mammalian cells [32,105,106], but their identification in plants is still awaited. The mechanism underlying NMD suppression during plant attack by pathogens is still not clear. There is currently a controversy about whether P. syringae infection downregulates UPF1 and UPF3 expression [85,87]. Further study will be necessary to fully understand the basis of NMD inhibition under this condition. It will also be very interesting to determine whether, in addition to pathogen attack, other environmental or developmental cues adjust the efficiency of the NMD process itself and, thereby, modify entire physiological programs (see Outstanding Questions). Acknowledgments The author thanks Sharon Victor for text editing. This work was supported by the Israel Science Foundation (grant 199/09).

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