Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7

Eukaryotic mRNAs containing premature termination codons (PTCs) are degraded by a process known as nonsense-mediated mRNA decay (NMD). NMD has been suggested to require the recognition of PTC by an mRNA surveillance complex containing UPF1/SMG-2. In multicellular organisms, UPF1/SMG-2 is a phosphoprotein, and its phosphorylation contributes to NMD. Here we show that phosphorylated hUPF1, the human ortholog of UPF1/SMG-2, forms a complex with human orthologs of the C. elegans NMD proteins SMG-5 and SMG-7. The complex also associates with protein phosphatase 2A (PP2A), resulting in dephosphorylation of hUPF1. Overexpression of hSMG-5 mutants that retain interaction with P-hUPF1 but which cannot induce its dephosphorylation impair NMD, suggesting that NMD requires P-hUPF1 dephosphorylation. We also show that P-hUPF1 forms distinct complexes containing different isoforms of hUPF3A. We propose that sequential phosphorylation and dephosphorylation of hUPF1 by hSMG-1 and PP2A, respectively, contribute to the remodeling of the mRNA surveillance complex.     

PTC下游翻译的重新起始导致NMD途径的逃逸

无义突变介导的mRNA降解（nonsense mediated mRNA decay, NMD）途径是真核生物体内一种重要的mRNA监督质控机制, 它降解含有由无义突变、错误剪接、移码突变等产生的提前终止翻译密码子（premature translation termination codon, PTC）的mRNA, 从而防止这种mRNA翻译产生的截短型蛋白质对机体造成的伤害. 研究发现, 一些含有PTC的mRNA发生了NMD途径逃逸, 但具体机制仍不清楚.本研究将成视网膜细胞瘤基因1 (retinoblastoma gene 1, RB1)作为NMD途径的靶基因, 构建mini-RB1基因,包括外显子1～14(cDNA)、内含子14 外显子15 内含子15和外显子16～27(cDNA) 的三部分序列, 将其构建到真核表达载体pcDNA 3.1(-)中.根据人类基因组突变数据库选择3个突变位点W99X、G310X和R467X, 构建相应无义突变体.分别将mini RB1基因野生型和无义突变体转入HeLa细胞进行表达.用qRT-PCR检测发现, W99X无义突变体与野生型相比mRNA的水平无显著差异.为了进一步证实mini- RB1（W99X）发生了NMD逃逸, 利用NMD途径抑制剂放线菌酮和转录抑制剂放线菌素D, 分别处理转入野生型的mini RB1基因及其无义突变体mini-RB1（W99X）的哺乳动物细胞, 发现mini-RB1基因无义突变体的mRNA水平与野生型无明显差异, 说明含有W99X无义突变的mini-RB1基因的mRNA发生了NMD逃逸.Western印迹检测mini-RB1基因的蛋白质表达发现, 在无义突变位点W99X下游重新起始了蛋白质的翻译, 因此,PTC下游蛋白质翻译的重新起始可能是导致无义mRNA逃逸NMD途径监控的主要原因.     

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

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that recognizes and degrades mRNAs with premature termination codons (PTCs). In yeast, PTC-containing mRNAs are targeted to processing bodies (P-bodies), and yeast strains expressing an ATPase defective Upf1p mutant accumulate P-bodies. Here we show that in human cells, an ATPase-deficient UPF1 mutant and a fraction of UPF2 and UPF3b accumulate in cytoplasmic foci that co-localize with P-bodies. Depletion of the P-body component Ge-1, which prevents formation of microscopically detectable P-bodies, also impairs the localization of mutant UPF1, UPF2, and UPF3b in cytoplasmic foci. However, the accumulation of the ATPase-deficient UPF1 mutant in P-bodies is independent of UPF2, UPF3b, or SMG1, and the ATPase-deficient UPF1 mutant can localize into the P-bodies independent of its phosphorylation status. Most importantly, disruption of P-bodies by depletion of Ge-1 affects neither the mRNA levels of PTC-containing reporter genes nor endogenous NMD substrates. Consistent with the recently reported decapping-independent SMG6-mediated endonucleolytic decay of human nonsense mRNAs, our results imply that microscopically detectable P-bodies are not required for mammalian NMD.     

Targeting of aberrant mRNAs to cytoplasmic processing bodies

In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.     

真核生物无义介导的mRNA降解与肿瘤关系的研究进展

研究发现任何错误剪接、移码、插入等基因突变都可能引入含有提前终止密码子(premature termination codon,PTC)的转录产物,将导致翻译提前终止而产生无生物活性甚至毒害性截短蛋白(truncated proteins)。而无义介导的mRNA的降解(nonsensemediated m RNA decay,NMD)作用机制在基因转录及转录后加工过程中选择性地迅速降解含有提前终止密码子的mRNA,避免产生对细胞正常生理功能有害的截短蛋白,真核生物NMD是转录后m RNA监控的重要环节。肿瘤的发生发展与相应基因的表达有关,NMD可以降解含有PTC的mRNA,学者们认为抑制NMD后肿瘤中某些基因的表达上调,而上调的基因或许在肿瘤的发生发展中其抑癌基因的作用,故学者们抑制肿瘤中NMD后进行测序筛选发生无义突变的抑癌基因。     

NMD逃逸机制及其在疾病治疗中的应用

无义介导的mRNA降解(nonsense-mediated mRNA decay, NMD)是指在病理或正常生理情况下mRNA上出现了提前终止密码子(premature termination codon, PTC),从而导致mRNA降解。它是一种广泛存在的mRNA质量监控机制。近年来,在多种疾病中发现某些PTC并未触发NMD,这种现象被称为NMD逃逸(NMD escape),然而其确切机制尚不十分清楚。目前公认的两个学说为:(1) PTC通读,即蛋白的翻译可以顺利通过PTC直至正常的终止密码子,产生全长蛋白;(2)翻译的重新启动,即蛋白翻译在PTC下游的潜在起始点重新开始直至终止密码子,产生N端截短蛋白。目前,通过利用PTC通读,越来越多的药物或小分子已被成功用于无义变异相关疾病的治疗。本文主要综述了NMD逃逸的机制及其在疾病治疗中的应用和进展,以期为进一步了解NMD逃逸及其相关应用概况提供参考。     

Nonsense-mediated mRNA decay (NMD) silences the accumulation of aberrant trypsin proteinase inhibitor mRNA in Nicotiana attenuata

In eukaryotes, genes carrying premature termination codons (PTCs) are often associated with decreased mRNA levels compared with their counterparts without PTCs. PTC-harboring mRNA is rapidly degraded through the nonsense-mediated mRNA decay (NMD) pathway to prevent the accumulation of potentially detrimental truncated proteins. In a native ecotype of Nicotiana attenuata collected from Arizona (AZ), the mRNA levels of a trypsin proteinase inhibitor ( TPI ) gene are substantially lower than in plants collected from Utah (UT). Cloning the AZ TPI gene revealed a 6 bp deletion mutation in exon 2 resulting in a PTC and decreased mRNA levels through NMD. Silencing UPF1 , 2 and 3 in N. attenuata AZ plants by virus-induced gene silencing (VIGS) enhanced the levels of PTC-harboring TPI mRNA, demonstrating a conserved role for UPF genes in plants. Furthermore, using cell suspension cultures that express variants of the TPI construct, we demonstrate that both intron-containing and intronless genes are subject to NMD in plants; unlike PTCs in mammals, PTCs downstream of introns activate NMD in plants. However, when a PTC is only 4 bp upstream of an intron, the NMD surveillance mechanism is abrogated. We also demonstrate that, in an intronless TPI gene, a PTC located at the beginning or the end of the coding sequence triggers NMD less efficiently than do PTCs located at the middle of the coding sequence. Taken together, these results highlight the complexity of the NMD activation mechanisms in plants.     

A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD substrates can be degraded by different routes that all require phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of the three known NMD factors thought to be recruited to nonsense mRNAs via an interaction with P-UPF1, leading to eventual mRNA degradation. By artificial tethering of SMG6 and mutants thereof to a reporter mRNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 to reduce reporter mRNA levels. Using in vivo and in vitro approaches, we further document that SMG6 and the unique stalk region of the UPF1 helicase domain, along with a contribution from the SQ domain, form a novel interaction and we also show that this region of the UPF1 helicase domain is critical for SMG6 function and NMD. Our results show that this interaction is required for NMD and for the capability of tethered SMG6 to degrade its bound RNA, suggesting that it contributes to the intricate regulation of UPF1 and SMG6 enzymatic activities.     

N- and C-terminal Upf1 phosphorylations create binding platforms for SMG-6 and SMG-5:SMG-7 during NMD

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature termination codons (PTCs). SMG-1-mediated Upf1 phosphorylation takes place in the decay inducing complex (DECID), which contains a ribosome, release factors, Upf1, SMG-1, an exon junction complex (EJC) and a PTC-mRNA. However, the significance and the consequence of Upf1 phosphorylation remain to be clarified. Here, we demonstrate that SMG-6 binds to a newly identified phosphorylation site in Upf1 at N-terminal threonine 28, whereas the SMG-5:SMG-7 complex binds to phosphorylated serine 1096 of Upf1. In addition, the binding of the SMG-5:SMG-7 complex to Upf1 resulted in the dissociation of the ribosome and release factors from the DECID complex. Importantly, the simultaneous binding of both the SMG-5:SMG-7 complex and SMG-6 to phospho-Upf1 are required for both NMD and Upf1 dissociation from mRNA. Thus, the SMG-1-mediated phosphorylation of Upf1 creates a binding platforms for the SMG-5:SMG-7 complex and for SMG-6, and triggers sequential remodeling of the mRNA surveillance complex for NMD induction and recycling of the ribosome, release factors and NMD factors.     

CK2-mediated TEL2 phosphorylation augments nonsense-mediated mRNA decay (NMD) by increase of SMG1 stability

Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism that degrades a premature-termination codon (PTC)-containing mRNA. During mammalian NMD, SMG1 and UPF1, key proteins in NMD, join at a PTC and form an SMG1–UPF1–eRF1–eRF3 (SURF) complex by binding UPF1 to eRF3 after PTC-recognition by the translating ribosome. Subsequently, UPF1 is phosphorylated after UPF1–SMG1 moves onto the downstream exon junction complex (EJC). However, the cellular events that induce UPF1 and SMG1 complex formation and increase NMD efficiency before PTC recognition remain unclear. Here, we show that telomere-maintenance 2 (TEL2) phosphorylation by casein-kinase 2 (CK2) increases SMG1 stability, which increases UPF1 phosphorylation and, ultimately, augments NMD. Inhibition of CK2 activity or downregulation of TEL2 impairs NMD. Intriguingly, loss of TEL2 phosphorylation reduces UPF1-bound PTC-containing mRNA and the formation of the SMG1–UPF1 complex. Thus, our results identify a new function of CK2-mediated TEL2 phosphorylation in a mammalian NMD.     

Testing the faux-UTR model for NMD: analysis of Upf1p and Pab1p competition for binding to eRF3/Sup35p

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that accelerates the degradation of mRNAs containing premature translation termination codons. This quality control pathway depends on the NMD-specific factors, Upf1p, Upf2p/Nmd2p, and Upf3p, as well as the two release factors, eRF1 and eRF3 (respectively designated Sup45p and Sup35p in yeast). NMD activation is also enabled by the absence of the poly(A)-binding protein, Pab1p, downstream of a termination event. Since Sup35p interacts with both Upf1p and Pab1p we considered the possibility that differential binding of the latter factors to Sup35p may be a critical determinant of NMD sensitivity for an mRNA. Here we describe three approaches to assess this hypothesis. First, we tethered fragments or mutant forms of Sup35p downstream of a premature termination codon in a mini-pgk1 nonsense-containing mRNA and showed that the inhibition of NMD by tethered Sup35p does not depend on the domain necessary for the recruitment of Pab1p. Second, we examined the Sup35p interaction properties of Upf1p and Pab1p in vitro and showed that these two proteins bind differentially to Sup35p. Finally, we examined competitive binding between the three proteins and observed that Upf1p inhibits Pab1p binding to Sup35p whereas the interaction between Upf1p and Sup35p is relatively unaffected by Pab1p. These data indicate that the binding of Upf1p and Pab1p to Sup35p may be more complex than anticipated and that NMD activation could involve more than just simple competition between these factors. We conclude that activation of NMD at a premature termination codon is not solely based on the absence of Pab1p and suggest that a specific recruitment step must commit Upf1p to the process and Upf1p-associated mRNAs to NMD.     

The multiple lives of NMD factors: balancing roles in gene and genome regulation

Nonsense-mediated mRNA decay (NMD) largely functions to ensure the quality of gene expression. However, NMD is also crucial to regulating appropriate expression levels for certain genes and for maintaining genome stability. Furthermore, just as NMD serves cells in multiple ways, so do its constituent proteins. Recent studies have clarified that UPF and SMG proteins, which were originally discovered to function in NMD, also have roles in other pathways, including specialized pathways of mRNA decay, DNA synthesis and cell-cycle progression, and the maintenance of telomeres. These findings suggest a delicate balance of metabolic events - some not obviously related to NMD - that can be influenced by the cellular abundance, location and activity of NMD factors and their binding partners.     

Context analysis of termination codons in mRNA that are recognized by plant NMD

The nonsense-mediated mRNA decay (NMD) system is an RNA surveillance system that degrades mRNAs possessing premature translation termination codons (PTCs). Although NMD factors are well conserved in eukaryotes, it is speculated that the contexts of those termination codons that are subject to NMD are different depending on the organism. Context analysis of termination codons that are recognized by the plant NMD system would clarify NMD target mRNAs in plants, and contribute to our understanding of its biological relevance in plants. In the present study we analyzed the positions of termination codons that were recognized as PTCs using an Agrobacterium transient expression assay, i.e. the accumulation of a series of plant mRNAs with nonsense mutations in different contexts was tested in plants. The results indicated that termination codons that are located distant from the mRNA 3' termini or >50 nucleotides upstream of the 3'-most exon-exon junction are recognized as substrates for NMD.