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Structure of the Nmd4-Upf1 complex supports conservation of the nonsense-mediated mRNA decay pathway between yeast and humans [1]

['Irène Barbarin-Bocahu', 'Laboratoire De Biologie Structurale De La Cellule', 'Bioc', 'Cnrs', 'Ecole Polytechnique', 'Institut Polytechnique De Paris', 'Palaiseau', 'Nathalie Ulryck', 'Amandine Rigobert', 'Nadia Ruiz Gutierrez']

Date: 2024-10

The nonsense-mediated mRNA decay (NMD) pathway clears eukaryotic cells of mRNAs containing premature termination codons (PTCs) or normal stop codons located in specific contexts. It therefore plays an important role in gene expression regulation. The precise molecular mechanism of the NMD pathway has long been considered to differ substantially from yeast to metazoa, despite the involvement of universally conserved factors such as the central ATP-dependent RNA-helicase Upf1. Here, we describe the crystal structure of the yeast Upf1 bound to its recently identified but yet uncharacterized partner Nmd4, show that Nmd4 stimulates Upf1 ATPase activity and that this interaction contributes to the elimination of NMD substrates. We also demonstrate that a region of Nmd4 critical for the interaction with Upf1 in yeast is conserved in the metazoan SMG6 protein, another major NMD factor. We show that this conserved region is involved in the interaction of SMG6 with UPF1 and that mutations in this region affect the levels of endogenous human NMD substrates. Our results support the universal conservation of the NMD mechanism in eukaryotes.

Funding: This work was supported by the Centre National de la Recherche Scientifique (CNRS) to HLH and MG, the Ecole Normale Supérieure and the Institut National de la Santé et de la Recherche Médicale for HLH, Institut Pasteur to CS, the Agence Nationale pour la Recherche ANR-18-CE11-0003-01 to CS, ANR-18-CE11-0003-02 and ANR-22-CE12-0004 to HLH and ANR-18-CE11-0003-04 to MG, Ecole Polytechnique to MG, the French Ministère de l’Enseignement Supérieur et de la Recherche (MESR) to IBB/BG, Ecole doctorale Complexité du Vivant (ED 515, Sorbonne Université) for PhD funding of NRG and the Fondation ARC pour la Recherche sur le Cancer for PhD funding of NRG and IBB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we describe the crystal structure of the complex formed between Nmd4 and the Upf1 helicase domain of S. cerevisiae. This structure reveals that Nmd4 interacts with Upf1 helicase core through a conserved C-terminal arm, an interaction that is important for the NMD-activating role of Nmd4 in yeast. We also reveal that Nmd4 activates the ATPase activity of Upf1 and increases its affinity for RNA. Interestingly, we identify a region similar to the C-terminal arm of Nmd4 in SMG6 proteins in metazoans. We show that this region is important for the previously described phospho-independent interaction of the human SMG6 protein with the helicase domain of UPF1, as well as for the optimal degradation of NMD substrates in human cells. These results suggest that, in addition to the endonuclease function described for SMG6, which appears to have been lost in Nmd4, the interaction of this protein with UPF1 may itself be important for NMD through its impact on the RNA helicase properties of this central NMD factor.

UPF1 is made of 2 structured domains: a CH-domain known to interact with UPF2 and an ATP-dependent RNA helicase domain (HD). Both domains are surrounded by N- and C-terminal extensions, which are predicted to be unfolded. Ebs1 is predicted to contain a 14-3-3 domain [ 46 ]. Such domain is generally involved in the recognition of phosphoserine or phosphothreonine residues [ 47 ]. Ebs1 acts as a translation inhibitor and promotes NMD [ 46 , 48 ] but no other studies have focused on this protein. Nmd4 is composed of a PIN domain (for PilT N-terminus) [ 49 ] and interacts directly with the helicase domain of Upf1 [ 45 ]. Although Nmd4 is not an essential NMD factor, it plays an important role for NMD under specific conditions such as the presence of a Upf1 protein lacking its CH domain [ 45 ]. Interestingly, yeast Ebs1 could be a functional homolog of metazoan SMG5, SMG6, and SMG7 proteins, all of which include a 14-3-3 domain [ 31 , 46 , 50 , 51 ], while the PIN domain of Nmd4 is structurally similar to the PIN domains of SMG5 and SMG6 [ 34 , 49 ]. Overall, these studies suggest that a global mechanism is conserved from yeast to human for the NMD pathway, with some elaborate branches (such as the role of the EJC) in metazoans. The truly conserved features of such a universally conserved molecular mechanism of NMD are unclear.

Recent publications have provided some nuances to these initial models, calling for further studies aimed at clarifying the molecular mechanism of NMD. First, S. cerevisiae Pab1 is dispensable to discriminate between normal and premature stop codons [ 38 ]. Second, the EJC now emerges as an enhancer of NMD even if its interaction with UPF3 is not mandatory for this process [ 24 , 39 , 40 ]. This can be explained for example, by the role of EJC in enhancing translation [ 41 ], which, in turn can facilitate the detection and degradation of NMD substrates. Third, human UPF3B interacts directly with UPF1 as well as with eRF1 and eRF3a, unlike UPF1 [ 42 ], in opposition with the SURF model. Fourth, the SMG6-dependent RNA degradation pathway in NMD depends on the SMG5/7 complex, as the absence of the latter prevents endonucleolytic cleavage of mRNA by SMG6 [ 43 ]. This is in line with a previous observation showing that SMG6 and SMG5-7 complexes target essentially the same genes [ 44 ]. Finally, using fast affinity purification coupled to quantitative mass spectrometry [ 45 ], we recently showed that in the yeast S. cerevisiae, Upf1 is part of 2 distinct and mutually exclusive complexes: the Upf1-2/3 complex and the Upf1-decapping complex. The latter encompasses Upf1, the Dcp1, Dcp2, and Edc3 decapping factors as well as 2 largely uncharacterized proteins, Nmd4 and Ebs1. These 2 additional yeast NMD co-factors show similarities with metazoan SMG6 and SMG5/7, both at the level of their sequence and in term of their interactions with Upf1.

NMD occurs through successive steps aimed at recognizing a stop codon as premature and then at subsequently degrading the aberrant mRNAs. The precise molecular mechanism of the NMD pathway is still unclear and remains controversial. Many lines of evidence supported the existence of several possible scenarios to recruit different factors to trigger the decay of NMD substrates [ 21 ]. Two models, which are not mutually exclusive, are currently proposed for NMD. Both rely on the involvement of 3 particularly important factors, UPF1, UPF2, and UPF3, in various eukaryotic model systems. The first model, the 3′-faux UTR model posits that for mRNAs with long 3′ UTRs, a long spatial distance between a stop codon and the mRNA poly(A) tail destabilizes NMD substrates. Indeed, it would prevent the physical interaction between the eRF1-eRF3 translation termination complex recognizing a stop codon in the A-site of the ribosome and the poly(A)-binding protein (Pab1 or PABP in Saccharomyces cerevisiae and human, respectively) bound to the 3′ poly(A) tail [ 22 – 24 ]. In this context, the UPF1 RNA helicase interacts with the eRF1-eRF3 complex bound to terminating ribosomes together with the UPF2 and UPF3 proteins [ 25 , 26 ], thereby tagging this stop codon as a PTC, and subsequently recruits RNA decay factors [ 27 ]. Alternatively, for mRNAs harboring PTCs more than 50 to 55 nucleotides upstream of an exon-exon junction [ 28 ], the presence of the exon junction complex (EJC) bound downstream of the PTC triggers mRNA elimination by the NMD pathway. Indeed, the recognition of PTCs by the SURF complex (for SMG-1/UPF1/eRF1-3 complex) allows the interaction of this complex with the UPF2 and UPF3 proteins bound to the EJC, thereby leading to the formation of the DECID complex (for Decay Inducing Complex) and the subsequent phosphorylation of UPF1 [ 26 ]. The phosphorylated UPF1 can then recruit the SMG5/SMG7 heterodimer, which subsequently interacts with the CCR4-NOT deadenylation complex and the decapping factors to eliminate aberrant mRNAs [ 29 , 30 ]. Alternatively, the SMG6 endonuclease can be recruited via its interaction with the EJC or through phospho-dependent and phospho-independent interactions with UPF1 [ 31 – 37 ].

In eukaryotes, several cytoplasmic surveillance pathways monitor the quality of translated mRNAs to prevent ribosomes stalling on faulty mRNAs or the synthesis of aberrant proteins [ 1 – 4 ]. Among these, the nonsense-mediated mRNA decay (NMD) pathway is responsible for the rapid detection and degradation of translated mRNAs harboring premature termination codons (PTCs), which can arise from mutations, transcription, or processing errors as well as alternative or defective splicing events [ 3 – 6 ]. The NMD mechanism also targets mRNAs, small nucleolar RNAs (snoRNAs), and long noncoding RNAs (lncRNAs) carrying normal stop codons located in a specific context (short upstream open reading frame or uORF, long 3′ UTRs, low translational efficiency or exon-exon junction located downstream of a stop codon [ 3 , 4 , 7 – 13 ]). NMD hence plays an important role in gene expression regulation and could monitor that the correct reading frame is scanned by the ribosome since most out-of-frame translation events would result in the detection of a premature stop codon by the ribosome [ 14 ]. The NMD pathway is commonly considered as a double-edged sword protecting cells from the synthesis of potentially harmful truncated proteins but also inhibiting the synthesis of partially or fully functional truncated proteins. Thereby, NMD is considered to be involved in many forms of cancers [ 15 , 16 ], in about 20% of all genetic diseases caused by nonsense mutations such as neurodegenerative diseases [ 17 , 18 ] and also to be a major defense mechanism against viral infections [ 19 , 20 ].

Results

Nmd4 stimulates Upf1 ATPase activity In the Nmd4/Upf1-HD interface, the Nmd4 « arm » accounts for around 75% of the interface area, while its PIN domain represents only 25%. This led us to investigate the importance of each Nmd4 domain for the interaction with Upf1-HD. Using in vitro His-pull down, we observed that Upf1-HD interacts specifically with full-length Nmd4 or with the « arm » region alone, but not with the PIN domain (S5A Fig). This interaction was further quantified by isothermal titration calorimetry (ITC) experiments showing that full-length Nmd4 or Nmd4 « arm » interacted with Upf1-HD with a Kd of 2.1 μm and 1.96 μm, respectively, whereas no interaction was detected between the PIN domain of Nmd4 and Upf1-HD in our experimental conditions (Table 1 and Fig 2A). In parallel, we performed co-immunoprecipitation experiments in vivo. Unfortunately, an HA-tag version of the « arm » was not stably expressed in S. cerevisiae yeast. However, unlike the full-length Nmd4 protein, the single PIN domain did not co-purify with TAP-tagged Upf1 (S5B Fig). Overall, these results indicate that the « arm » region of Nmd4 is sufficient and necessary for a stable Nmd4/Upf1 interaction. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Nmd4 influences Upf1 ATPase activity. (A) Characterization of the interaction between His 6 -ZZ-Nmd4 full-length protein (upper left panel), His 6 -ZZ-Nmd4 « arm » (upper right panel), His 6 -ZZ-Nmd4 PIN domain (lower left panel) or the His 6 -ZZ tag (lower right panel), and Upf1-HD by ITC. Upper panel: ITC data obtained by injecting the different Nmd4 constructs or the His 6 -ZZ tag into Upf1-HD. Lower panel: Fitting of the binding curves using a single binding site model. Three biologically independent experiments were performed and a representative image is shown. The data underlying this figure can be found in S1 Data. (B) Nmd4 stimulates Upf1-HD ATPase activity. Purified Upf1-HD alone or in combination with full-length or fragments of Nmd4 or His-ZZ (control) was used in a 32P release assay, as a readout of the ATPase activity. The relative activity (indicated in percent) corresponds to the activity measured in a given condition (different proteins) normalized to the activity of Upf1-HD alone after 30 min of reaction at 30°C. The relative activity for each condition after 30 min is indicated on the right of the different curves. The data underlying this figure can be found in S2 Data. HD, helicase domain; ITC, isothermal titration calorimetry. https://doi.org/10.1371/journal.pbio.3002821.g002 PPT PowerPoint slide

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TIFF original image Download: Table 1. Thermodynamics parameters for the interaction of Upf1-HD with Nmd4 or RNA as determined by ITC. https://doi.org/10.1371/journal.pbio.3002821.t001 As the role of the interaction between Nmd4 and Upf1 is still largely unknown, we analyzed its effect on Upf1 ATPase activity. We observed that, in vitro, the basal ATPase activity of the yeast Upf1-HD was strongly stimulated upon incubation with the full-length Nmd4 protein (Fig 2B). The « arm » of Nmd4, which is mandatory for the Nmd4/Upf1-HD interaction, also enhanced Upf1-HD ATPase activity, but to a much lower extent. Surprisingly, the PIN domain alone also stimulated Upf1-HD helicase activity as effectively as the « arm ». The various Nmd4 constructs alone had no ATPase activity. Similarly, the His-ZZ tag used to express the various Nmd4 construct did not activate significantly Upf1-HD activity. This indicates that the observed increase in enzymatic activity is specific to the binding of Nmd4 to Upf1-HD (Fig 2B). Altogether, this strongly suggests that the PIN domain and the « arm » act synergistically to enhance Upf1 ATPase activity, but also that a physical interaction between Upf1-HD and the PIN domain might exist in vitro. We were unable to detect such an interaction using our different interaction assays (pull-down and ITC), which are optimal for studying interactions with dissociation constants (Kd) in the nanoM to tens of microM range. We therefore assume that a transient low-affinity interaction (high Kd value not detected by our binding assays) exists between Upf1-HD and PIN Nmd4 and can only be detected by highly sensitive assays such as our radioactivity-based ATPase assay, which was performed with a 20-fold molar excess of PIN Nmd4 domain over Upf1-HD. This also indicates that Nmd4 has a long-range effect on Upf1 helicase activity, since no Nmd4 residue interacts in the vicinity of the Upf1 ATPase active site. This could be due to the extended interaction of Nmd4 with 3 important regions (RecA1, stalk, and RecA2) of Upf1-HD. To investigate the influence of Nmd4 on another Upf1-HD activity, namely RNA binding, we determined the Kd of Upf1-HD for a poly(U) 30 oligonucleotide in the absence or in the presence of Nmd4 by ITC. First, Upf1-HD, but also surprisingly Nmd4 alone, interacted with a poly(U) 30 RNA with Kd values of 1.03 μm and 8.1 μm, respectively (Table 1 and S6 Fig). Interestingly, the Nmd4/Upf1-HD complex has a 2.3-fold lower Kd value for this RNA than Upf1-HD (Kd = 0.44 μm). Whether this increase in affinity is due to a synergistic effect between both proteins or to an allosteric stimulation of one partner on the RNA binding property of the second partner remains to be clarified.

The Nmd4/Upf1-HD interaction is important for NMD in vivo Detailed analysis of the crystal structure of the Nmd4/Upf1-HD complex revealed that 2 strictly conserved residues, located in the « arm » region of Nmd4, were of particular interest. The side chain of R210 forms 3 hydrogen bonds with the carbonyl group of F368 as well as with the hydroxyl group of S374 from Upf1-HD, while the W216 side chain stacks on the helix α1 and in particular on G243 of the Upf1-HD stalk region (Fig 3A and 3B and S2 Table). To investigate the importance of these residues for complex formation, we generated 2 single point mutants (R210E to introduce charge inversion and W216A to disrupt several van der Waals interactions) as well as the double mutant (R210E/W216A; referred to as M2). Similarly, we mutated Upf1-HD G243 and G377 to arginines to induce steric hindrance with Nmd4 as they lie close to residues W216 and R210 of Nmd4, respectively (Fig 3A and 3B). Again, 2 single point mutants (G243R or G377R) as well as the double mutant (G243R/G377R, referred to as DM) were generated. During the purification process, all mutants behaved similarly to the wild-type proteins, indicating that the overall structure of the proteins were not affected by the mutations (S7A and S7B Fig). All Upf1-HD mutants lost their ability to interact in vitro with Nmd4 compared with the wild-type protein as demonstrated by the specific enrichment of Upf1-HD WT but not of the mutants by CBP-His 6 -ZZ-Nmd4 (compare lane 7 to lanes 8 to 10; Fig 3C). Thus, the substitution of glycine by a large, positively charged arginine side chain strongly interferes with the formation of the Nmd4/Upf1-HD complex. Similarly, we observed that each Upf1 single point mutant (G243R or G377R) associated much more weakly with Nmd4-HA in vivo (Fig 3D). We then studied the effect of these mutants on the in vivo stability of 2 endogenous (DAL7 and pre-L28) yeast NMD substrates in a specific context where Nmd4 is known to be important for NMD [45]. The expression of a Upf1 protein lacking the CH domain (hereafter referred to as Upf1-HD-Ct) only partially complemented the UPF1 deletion compared with ectopic expression of the full-length protein (pUpf1; Figs 3E and S8; [45]). Importantly, the complementation by Upf1-HD-Ct is further reduced in the upf1Δ/nmd4Δ double mutant. In the presence of the Upf1-HD-Ct construct, the levels of DAL7 and pre-L28 NMD substrates were similar in the upf1Δ strains lacking Nmd4 or upon expression of Upf1-HD-Ct G243R or G377R point mutants in the upf1Δ or upf1Δ/nmd4Δ strains. Altogether, this indicates that the interaction between Nmd4 and Upf1 is important for NMD in the absence of the Upf1 CH domain, i.e., when the efficient recruitment of Upf2 and/or decapping factors is impaired for instance [27,53]. PPT PowerPoint slide

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TIFF original image Download: Fig 3. The interaction of Nmd4 with Upf1 is important for NMD. (A) Zoom-in on the interaction network formed by the side chain of Arg210 from Nmd4. Hydrogen bonds are depicted as black dashed lines. The Cα atom of Gly377 from Upf1-HD is shown as a sphere. (B) Zoom-in on the side chain of Trp216 from Nmd4 lying on helix α1 from Upf1-HD stalk region. The Cα atom of Gly243 from Upf1-HD is shown as a sphere. (C) CBP-pull-down experiment showing that the Upf1-HD single mutants (G243R or G377R) or the double mutant (DM; G243R/G377R) affect the binding to CBP-tagged Nmd4. Input and eluate from calmodulin sepharose beads (IP) samples were analyzed on 12% SDS/PAGE and Coomassie blue staining. As controls, each Upf1-HD variant was incubated with the CBP-His 6 -ZZ tag, revealing some weak nonspecific retention of the various Upf1-HD proteins on the calmodulin sepharose beads. Degradation products enriched on calmodulin sepharose beads are indicated by an asterisk (*). Three biologically independent experiments were performed and a representative image is shown. (D) Single point mutations in full-length Upf1 disrupt its interaction with Nmd4 in vivo. Co-purification of Nmd4-HA with TAP-Upf1-FL, TAP-Upf1-FL-G243R, and TAP-Upf1-FL-G377R was evaluated by immunoblot. A negative control experiment used the N-terminal region of Upf1 (TAP-Upf1-CH, encompassing residues 1 to 208) known to be unable to interact with Nmd4 [45]. (E) Upf1 deficient for binding to Nmd4 is impaired for its NMD role. A upf1Δ strain was transformed with plasmids expressing N-terminal tagged full-length Upf1 (pUpf1), to restore NMD, a truncated WT version of the protein lacking the CH domain (UPF1-HD-Ct), which can partially complement NMD, or UPF1-HD-Ct single point mutants (G243R or G377R) that are unable to interact with Nmd4. A double mutant upf1Δ/nmd4Δ strain shows an exacerbated NMD defect phenotype, which is not rescued by the expression of UPF1-HD-Ct single point mutants (G243R or G377R) that are unable to interact with Nmd4. Two natural NMD substrates, the uORF containing DAL7 transcript (left) and the unspliced pre-mRNA for the ribosomal protein Rpl28 (right) were used to estimate NMD efficiency and their levels were measured by RT-qPCR. The obtained values were normalized for total RNA amounts using PGK1 and spliced RPL28 transcripts and all the results were adjusted to the situation in which full-length Upf1 was expressed (marked “pUpf1”). The experiments were repeated at least 3 times and the individual results are indicated as dots. Error bars represent standard deviation. The p-values of t tests (Welch variant with continuity correction) for comparisons between results are indicated. The data underlying this figure can be found in S3 Data. (F) Pull-down experiment showing that the His 6 -ZZ-Nmd4 single point mutants (R210E or W216A) or the double point mutant (M2; R210E/W216A) affect the binding to Upf1. Input and eluate from NiNTA magnetic beads samples were analyzed on 12% SDS/PAGE and Coomassie blue staining. As controls, Upf1-HD was incubated with the His 6 -ZZ tag. Three biologically independent experiments were performed and a representative image is shown. (G) The Nmd4 mutants are less potent activators of Upf1-HD ATPase activity than WT Nmd4. The assay was similar to that presented in Fig 2B. The data underlying this figure can be found in S4 Data. HD, helicase domain; NMD, nonsense-mediated mRNA decay; uORF, upstream open reading frame. https://doi.org/10.1371/journal.pbio.3002821.g003 We also observed that the various Nmd4 mutants (R210E, W216A, and M2) were strongly affected in their ability to interact with Upf1-HD compared with the wild-type Nmd4 protein in vitro (Fig 3F), confirming the role of the Nmd4 « arm » in the interaction with Upf1-HD. Consistent with these observations, these 3 Nmd4 mutants did not stimulate Upf1 ATPase activity as efficiently as the wild-type Nmd4 protein (Fig 3G). Overall, these experiments show that the interaction formed between the Nmd4 « arm » and Upf1-HD contributes to Upf1 function in NMD, and in particular to its ATPase activity.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002821

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