Y14 and hUpf3b form an NMD-activating complex

Messenger RNAs with premature translation termination codons (PTCs) are degraded by nonsense-mediated mRNA decay (NMD). In mammals, PTCs are discriminated from physiological stop codons by a process thought to involve the splicing-dependent deposition of an exon junction complex (EJC), EJC-mediated recruitment of Upf3, and Upf2 binding to the N terminus of Upf3. Here, we identify a conserved domain of hUpf3b that mediates an interaction with the EJC protein Y14. Tethered function analysis shows that the Y14/hUpf3b interaction is essential for NMD, while surprisingly the interaction between hUpf3b and hUpf2 is not. Nonetheless, hUpf2 is necessary for NMD mediated by tethered Y14. RNAi-induced knockdown and Y14 repletion of siRNA-treated cells implicates Y14 in the degradation of beta-globin NS39 mRNA and demonstrates that Y14 is required for NMD induced by tethered hUpf3b. These results uncover a direct role of Y14 in NMD and suggest an unexpected hierarchy in the assembly of NMD complexes.     

Evidence that the Upf1-related molecular motor scans the 3'-UTR to ensure mRNA integrity

Upf1 is a highly conserved RNA helicase essential for nonsense-mediated mRNA decay (NMD), an mRNA quality-control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). For the activation of NMD, UPF1 interacts first with a translation-terminating ribosome and then with a downstream exon-junction complex (EJC), which is deposited at exon-exon junctions during splicing. Although the helicase activity of Upf1 is indispensable for NMD, its roles and substrates have yet to be fully elucidated. Here we show that stable RNA secondary structures between a PTC and a downstream exon-exon junction increase the levels of potential NMD substrates. We also demonstrate that a stable secondary structure within the 3'-untranslated region (UTR) induces the binding of Upf1 to mRNA in a translation-dependent manner and that the Upf1-related molecules are accumulated at the 5'-side of such a structure. Furthermore, we present evidence that the helicase activity of Upf1 is used to bridge the spatial gap between a translation-termination codon and a downstream exon-exon junction for the activation of NMD. Based on these findings, we propose a model that the Upf1-related molecular motor scans the 3'-UTR in the 5'-to-3' direction for the mRNA-binding factors including EJCs to ensure mRNA integrity.     

Communication with the exon-junction complex and activation of nonsense-mediated decay by human Upf proteins occur in the cytoplasm

The nonsense-mediated mRNA decay (NMD) pathway rids eukaryotic cells of mRNAs with premature termination codons. There is contradictory evidence as to whether mammalian NMD is a nuclear or a cytoplasmic process. Here, we show evidence that NMD in human cells occurs primarily, if not entirely, in the cytoplasm. Polypeptides designed to inhibit interactions between NMD factors specifically impede NMD when exogenously expressed in the cytoplasm. However, restricting the polypeptides to the nucleus strongly impairs their NMD-inhibitory function, even for those intended to inhibit interactions between the exon-junction complex (EJC) and hUpf3 proteins, which localize primarily in the nucleus. NMD substrates classified based on cell fractionation assays as "nucleus associated" or "cytoplasmic" are all inhibited in the same manner. Furthermore, retention of the NMD factor hUpf1 in the nucleus strongly impairs NMD. These observations suggest that the hUpf complex communicates with the EJC and triggers NMD in the cytoplasm.     

Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay

Decapping is a key step in general and regulated mRNA decay. In Saccharomyces cerevisiae it constitutes a rate-limiting step in the nonsense-mediated decay pathway that rids cells of mRNAs containing premature termination codons. Here two human decapping enzymes are identified, hDcp1a and hDcp2, as well as a homolog of hDcp1a, termed hDcp1b. Transiently expressed hDcp1a and hDcp2 proteins localize primarily to the cytoplasm and form a complex in human cell extracts. hDcp1a and hDcp2 copurify with decapping activity, an activity sensitive to mutation of critical hDcp residues. Importantly, coimmunoprecipitation assays demonstrate that hDcp1a and hDcp2 interact with the nonsense-mediated decay factor hUpf1, both in the presence and in the absence of the other hUpf proteins, hUpf2, hUpf3a, and hUpf3b. These data suggest that a human decapping complex may be recruited to mRNAs containing premature termination codons by the hUpf proteins.     

Nonsense mutations in close proximity to the initiation codon fail to trigger full nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs containing premature translation termination codons. In mammalian cells, a termination codon is ordinarily recognized as "premature" if it is located greater than 50-54 nucleotides 5' to the final exon-exon junction. We have described a set of naturally occurring human beta-globin gene mutations that apparently contradict this rule. The corresponding beta-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type beta-globin (beta(WT)) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between beta(WT) mRNA and beta-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; beta 39). Functional analyses of these mRNAs with 5'-proximal nonsense mutations demonstrate that their relative resistance to NMD does not reflect abnormal RNA splicing or translation re-initiation and is independent of promoter identity and erythroid specificity. Instead, the proximity of the nonsense codon to the translation initiation AUG constitutes a major determinant of NMD. Positioning a termination mutation at the 5' terminus of the coding region blunts mRNA destabilization, and this effect is dominant to the "50-54 nt boundary rule." These observations impact on current models of NMD.     

Functions of hUpf3a and hUpf3b in nonsense-mediated mRNA decay and translation

The exon-junction complex (EJC) components hUpf3a and hUpf3b serve a dual function: They promote nonsense-mediated mRNA decay (NMD), and they also regulate translation efficiency. Whether these two functions are interdependent or independent of each other is unknown. We characterized the function of the hUpf3 proteins in a lambdaN/boxB-based tethering system. Despite the high degree of sequence similarity between hUpf3b and hUpf3a, hUpf3a is much less active than hUpf3b to induce NMD and to stimulate translation. We show that induction of NMD by hUpf3 proteins requires interaction with Y14, Magoh, BTZ, and eIF4AIII. The protein region that mediates this interaction and discriminates between hUpf3a and hUpf3b in NMD function is located in the C-terminal domain and fully contained within a small sequence that is highly conserved in Upf3b but not Upf3a proteins. Stimulation of translation is independent of this interaction and is determined by other regions of the hUpf3 protein, indicating the presence of different downstream pathways of hUpf3 proteins either in NMD or in translation.     

Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4)

Nonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an important pathway used by all organisms that have been tested to degrade mRNAs that prematurely terminate translation and, as a consequence, eliminate the production of aberrant proteins that could be potentially harmful. In mammalian cells, NMD appears to involve splicing-dependent alterations to mRNA as well as ribosome-associated components of the translational apparatus. To date, human (h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its Saccharomyces cerevisiae orthologue that functions in both translation termination and NMD, has been the only factor shown to be required for NMD in mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid similarities. The existence of these orthologues provides evidence for a higher degree of evolutionary conservation of NMD than previously appreciated. Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4) derive from two genes, one of which is X-linked and both of which generate multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the domains required for the interactions. Furthermore, we find by using indirect immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei. The finding that hUpf3p-X is a shuttling protein provides additional indication that NMD has both nuclear and cytoplasmic components.     

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.     

Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay

mRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5' to the terminal exon-exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.     

New insights into the formation of active nonsense-mediated decay complexes

In the nonsense-mediated mRNA decay (NMD) pathway, an exon-junction protein complex (EJC) and hUpf proteins mediate rapid downregulation of aberrant mRNAs that terminate translation upstream of the last splice junction. Two EJC subunits, Y14 and RNPS1, have been proposed to act as a link between splicing and NMD by recruiting hUpf3 and the other hUpf proteins. New studies now present evidence that Y14 is directly involved in NMD, and that Y14 is required for hUpf3 activity. These findings suggest unforeseen intricacies in the formation of active NMD complexes.     

A new function for nonsense-mediated mRNA-decay factors

mRNAs often contain premature-termination (nonsense) codons as a result of mutations and RNA splicing errors. These nonsense codons cause rapid decay of the mRNAs that contain them, a phenomenon called nonsense-mediated mRNA decay (NMD). This response is thought to be a quality-control mechanism that protects cells from truncated dominant-negative proteins. Surprisingly, recent evidence strongly suggests that the NMD factors UPF1, UPF2, UPF3B, RNPS1, Y14 and MAGOH also promote translation of normal mRNAs in mammalian cells. This, along with an earlier discovery that NMD factors appear to dictate efficient translation termination, suggests that NMD factors do not merely function in RNA surveillance. These findings lead to the interesting question of why NMD factors evolved; are they for RNA-quality control or to promote efficient translation initiation and termination?     

A 3' UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus

Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation terminates within the coding region. In mammalian cells, this is presumably due to a downstream signal deposited during pre-mRNA splicing. In contrast, unspliced retroviral RNA undergoes NMD in chicken cells when premature termination codons (PTCs) are present in the gag gene. Surprisingly, deletion of a 401-nt 3' UTR sequence immediately downstream of the normal gag termination codon caused this termination event to be recognized as premature. We termed this 3' UTR region the Rous sarcoma virus (RSV) stability element (RSE). The RSE also stabilized the viral RNA when placed immediately downstream of a PTC in the gag gene. Deletion analysis of the RSE indicated a smaller functional element. We conclude that this 3' UTR sequence stabilizes termination codons in the RSV RNA, and termination codons not associated with such an RSE sequence undergo NMD.     

Nonsense-mediated decay of glutathione peroxidase 1 mRNA in the cytoplasm depends on intron position

mRNA for glutathione peroxidase 1 (GPx1) is subject to cytoplasmic nonsense-mediated decay (NMD) when the UGA selenocysteine (Sec) codon is recognized as nonsense. Here, we demonstrate by moving the sole intron of the GPx1 gene that either the Sec codon or a TAA codon in its place elicits NMD when located >/=59 bp but not 相似文献