Nonsense-mediated mRNA decay involves two distinct Upf1-bound complexes

Abstract

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to characterize the composition and dynamics of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Upf1-23 (Upf1, Upf2, Upf3) and Upf1-decappingUpf1-decapping contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association with RNA was partially dependent on Upf1-23 components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that NMD operates through conserved, successive Upf1-23 and Upf1-decapping complexes. This model can be extended to accommodate steps that are missing in yeast, to serve for further mechanistic studies of NMD in eukaryotes.

Keywords: NMD; Saccharomyces cerevisiae; RNA decay; affinity purification; quantitative mass spectrometry.

Figure 1. Enrichment analysis accurately describes Upf1‐associated proteins

1. A–C

   Positioning of Upf1, Upf2 and Upf3 in the abundance distribution of all yeast proteins, based on the data compiled by Ho et al (2018) (A). The distribution of abundance for the proteins identified by mass spectrometry in input (B), and purified samples (C), shows the expected bias for proteins present in high copy numbers. Light grey colour highlights the positions of proteins that were only identified in the Upf1‐TAP samples (six replicates) and not in the total extract (three replicates).

2. D

   Correlation of LTOP2‐based protein level estimates in a total yeast protein extract with published protein abundance data (Ho et al, 2018).

3. E

   Distribution of the intensity for the proteins quantified in association with Upf1 (LTOP2 score, log₂ scale). Positioning of Upf1 is highlighted as a white rectangle while Upf2 and Upf3 are highlighted in red. The dark grey line indicates the region of intensities corresponding to ribosomal proteins.

4. F

   Distribution of the protein enrichment values for Upf1‐associated proteins based on LTOP2 scores and known protein abundance in a total yeast extract. Upf1, Upf2 and Upf3 positions are also highlighted as white and red rectangles.

5. G

   A combination of the data presented in (E) and (F) as a scatter plot, to see both the amount and enrichment of proteins in Upf1‐TAP‐purified samples. The horizontal axis represents the LTOP2 score, with the vertical axis showing enrichment over input values. Both axes use log₂ transformed values.

Source data are available online for this figure.

Figure 2. Purification of NMD factors reveals two distinct complexes containing Upf1

1. A–F

   Enrichment values for proteins identified in tagged Upf1 (A), Upf2 (B), Upf3 (C), Nmd4 (D), Ebs1 (E) and Dcp1 (F) purifications. Tagged proteins are indicated in bold for each experiment. Black bars correspond to average enrichment values obtained in purifications done without RNase and orange bars with an RNase A and RNase T1 treatment. Error bars represent SD. Dots represent individual enrichment values for each protein in the six replicates for Upf1‐TAP, 3 for Upf1‐TAP and RNase, 6 for Upf2‐TAP, 3 for Upf2‐TAP and RNase, 5 for Upf3‐TAP, 3 for Upf3‐TAP and RNase, 4 for Nmd4‐TAP, 3 for Nmd4‐TAP and RNase, 3 for Ebs1‐TAP, with and without RNase and 3 for Dcp1‐TAP with or without RNase. Only proteins enriched by a factor of 16 or more in one of the purifications presented here are shown. For clarity, groups of related proteins were combined (the group of Lsm1 to Lsm7 is marked as “Lsm1‐7” and the values correspond to the mean of all the values found in purifications; CK2 group corresponds to Cka1, Cka2, Ckb1 and Ckb2).

2. G

   Immunoblot validation of Upf1‐TAP interactions with HA‐tagged Nmd4 and Ebs1. Control strains did not express Upf1‐TAP.

3. H

   Immunoblot validation of Upf1‐TAP interaction with Pat1‐HA, Lsm1‐HA and Edc3‐HA. The purification was performed with a mix of three strains, expressing Upf1‐TAP or not (control).

4. I

   Representation of binary interactions identified by our experiments: dashed lines correspond to interactions that are sensitive to RNase treatment; the arrows start at the tagged protein and indicate the enriched factor.

Source data are available online for this figure.

Figure EV1. Tagged proteins are functional for NMD

1. RNA from the indicated tagged strains and upf1Δ, as control, was quantified by reverse transcription followed by quantitative PCR for the unspliced precursor of RPL28 mRNA and for a normalization RNA, insensitive for NMD (RIM1). Mean values and standard deviations are depicted.

2. The stabilization of the endogenous NMD target pre‐RPL28 was tested in comparison with the mature form (RPL28) by northern blot using dsDNA probes and chemiluminescent detection.

Figure 3. Nmd4 and Ebs1 are the only Upf1‐decapping components that interact with Upf1 independent of the N‐terminal CH domain

1. A

   Upf1 fragments tested: FL is for the full‐length protein; HD‐Cter for the region containing the helicase domain and the C‐terminal part of Upf1 (208–971); CH for the N‐terminal domain (2–208) that contains the N‐terminal unstructured region and the cysteine/histidine‐rich domain; CH‐HD for a version a full‐length Upf1 lacking the C‐terminal region 854–971; and HD for the helicase domain of Upf1 alone (not to scale). N‐terminal TAP‐tagged Upf1 versions were expressed from a single‐copy vector under an inducible tetO7 promoter.

2. B–D

   Results of purifications using Upf1‐FL (B), Upf1‐CH (C) and Upf1‐Cter (D) as bait with the x‐axis showing the average enrichment value (log₂ scale) for Upf1‐decapping and Upf1‐23 components. The colour of the bars illustrates the treatment used during the purification, black without RNase and orange with an RNase treatment. Error bars represent SD. White dots correspond to enrichment values obtained in individual experiments.

3. E

   Comparison of the enrichment of Upf1, Nmd4 and Ebs1 in the purification of the Upf1 fragments. The Upf1 C‐terminal region (854–971) affected the association with Nmd4 or Ebs1 (Student t‐test with a one‐sided alternative hypothesis). We compared the enrichment of each of Ebs1 and Nmd4 between purifications of Upf1 fragments having this region (Upf1‐FL and Upf1‐HD‐Cter, six experiments) and purifications of Upf1 fragments lacking the extension (Upf1‐CH‐HD and Upf1‐HD, four experiments). The C‐terminal region had no effect on Nmd4 enrichment (P‐value ≈ 0.98), whereas there was significantly less associated Ebs1 on this small region (P‐value ≈ 0.0013).

4. F

   Western blot of input and eluates of Upf1 domains purification in a Nmd4‐HA strain. The band with the # might correspond to a dimer of Upf1‐CH, bands marked with a star correspond to residual signal with the anti‐HA antibodies (Nmd4). Fragments in the eluate have a smaller size because the protein A part of the tag was removed by digestion with the TEV protease. G6PDH served as a loading control in the input samples.

Source data are available online for this figure.

Figure EV2. N‐terminal and C‐terminal tagged Upf1 enrich similar sets of specific proteins

1. Estimation of the levels of overexpression for N‐terminal tagged Upf1 fragments, in comparison with chromosomally C‐terminal tagged protein. G6PDH was used as a loading control. Serial dilutions were used to test the ability of the immunoblot signal to estimate protein levels.

2. Average enrichment values for purifications done with chromosomally C‐terminal tagged Upf1 (x‐axis) and N‐terminally TAP‐tagged Upf1.

Figure 4. Nmd4 interaction with the Upf1‐decapping complex is direct and mediated by Upf1

1. Average enrichment of Upf1‐23 and Upf1‐decapping components in purified Upf1‐TAP in the presence (black bars) and absence (blue bars) of NMD4. Pink and dark blue vertical lines highlight proteins of the Upf1‐23 and Upf1‐decapping complexes, respectively. White dots correspond to individual enrichment values obtained in replicate experiments, 6 for Upf1‐TAP and 3 for the nmd4Δ condition. Error bars correspond to SD.

2. Average enrichment of Upf1‐decapping components in Nmd4‐TAP in the presence (black bars) and absence (blue bars) of UPF1. White dots correspond to individual enrichment values obtained in replicate experiments, 4 for Nmd4‐TAP and 2 for the upf1Δ condition. Error bars correspond to SD.

3. Schematics of Upf1 fragments used for the in vitro interaction assay; yUpf1‐HD is the helicase domain (220–851) of the yeast Upf1 protein, hUpf1‐HD is the helicase domain (295–914) of human Upf1 (not to scale).

4. CBP‐Nmd4 was mixed with hUPF1‐HD or yUPF1‐HD (all the proteins overexpressed in Escherichia coli and purified). Protein mixtures before (input, 20% of total) or after purification on calmodulin affinity beads were separated on 10% SDS–PAGE (w/v) acrylamide gels.

5. Upf1 interaction with a 30‐mer 3′ biotinylated RNA fragment was tested by mixing purified Upf1 helicase domain with Nmd4 and testing the fraction of recovered protein on streptavidin beads after washes with 150 and 300 mM NaCl containing buffer solution.

Source data are available online for this figure.

Figure 5. Nmd4 and Ebs1 are essential for NMD elicited by the overexpression of the helicase domain of Upf1

1. Schematic representation of the domain structure of Nmd4 and Ebs1 from Saccharomyces cerevisiae compared with human (h) Smg6, Smg5 and Smg7. 14‐3‐3, HHR and PIN domains were defined based on literature data (Fukuhara et al, 2005; Luke et al, 2007). Boundaries of the PIN domains were chosen based on a Mafft alignment of Smg5/6 of several species, while for Nmd4 the entire protein sequence was used. Drawing is not to scale. Identity percentages among the different domains of Smg proteins, Nmd4 and Ebs1, are indicated. Values represent per cent of identical residues as a fraction of all the aligned residues.

2. Scatter plot of the mean fold change of transcripts in nmd4∆ RNA‐Seq experiment against transcript mean fold change in ebs1∆ (Pearson correlation, r = 0.66, P‐value < 2 × 10⁻¹⁶). Black dots correspond to NMD substrate examples for which individual traces are shown in Fig EV3. Dashed lines represent boundaries of five bins of equal numbers of transcripts (2,046 transcripts or notable features per bin), with bin 5 containing the transcripts that were most increased in the mutant condition compared with wild type.

3. Percentage of transcripts affected by UPF1 deletion (increase by at least 1.4‐fold) among nmd4∆ bins, as defined in (B). Differences between percentages of upf1∆ affected transcripts in bin 4 and 5 and bin 3 and 4 were significant (binomial test, P < 10⁻²⁴).

4. Same as in (C), for ebs1∆. Differences between distribution of transcripts in bin n and bin n + 1 were significant (binomial test, P < 10⁻⁹).

5. NMD efficiency of WT, upf1∆ or double‐mutant strains complemented with Upf1‐FL, Upf1‐HD‐Cter or an empty plasmid. The NMD efficiency for each strain is based on reverse transcription followed by quantitative PCR for RPL28 pre‐mRNA. A wild‐type strain has 100% NMD efficiency and a upf1∆ strain has 0% NMD efficiency, by definition. Each value is an average of replicate experiments; 3 for the WT condition; 5 for upf1Δ; 3 for ufp1Δ/upf2Δ, upf1Δ/ufp3Δ and upf1Δ/ebs1Δ; and 4 for the upf1Δ/nmd4Δ condition. Error bars represent SD. Dots represent values obtained in the various replicate experiments.

6. Schematic drawing of an NMD reporter whose transcription can be repressed by doxycycline addition to the culture.

7. Changes in the steady‐state level of the reporter encoded HA‐tagged protein in upf1Δ, nmd4Δ, ebs1Δ and the nmd4Δ/ebs1Δ strains were estimated by immunoblot from three independent experiments, with one example shown.

8. RNA decay in a wild‐type and nmd4Δ/ebs1Δ strains was tested by reverse transcription quantitative PCR specific to the 5′ end of the reporter RNA at different times after transcription shut‐off. The quantifications represent mean RNA amounts and standard deviation of the average (three independent experiments).

Source data are available online for this figure.

Figure EV3. Deletion of NMD4 and EBS1 stabilize a set of transcripts that is also stabilized in the absence of UPF1

1. A–E

   Examples of NMD substrates sequencing profiles in WT, upf1∆, nmd4∆, ebs1∆ and nmd4∆/ebs1∆ experiments. NMD substrates belong to different classes, intron containing (RPL28; A and B, different scaling to show intron signal), uORF (DAL7, DAL2; C and D) and non‐coding RNA (SUT439; E). Profiles were normalized using the samples median counts.

2. F

   Scatter plot of transcript log₂ fold change in upf1∆ against ebs1∆. The dashed line represents the limit over which RNA was considered as stabilized, with a log₂ value of 0.5 (1.4 fold change).

Figure EV4. The helicase domain of Upf1 alone can destabilize RPL28 pre‐mRNA, an NMD substrate

1. Total RNA from wild‐type or a upf1Δ strain transformed with an empty plasmid (pControl) or plasmids expressing various Upf1 fragments (see Fig 3A) was tested by reverse transcription and quantitative PCR. The levels of RPL28 pre‐mRNA were normalized using an NMD‐insensitive transcript (RIM1), and an NMD efficiency score was calculated based on the difference between a wild‐type (100%) and the upf1Δ (0% NMD) strain.

2. Complementation of UPF1 deletion was tested in combination with the deletion of XRN1, and in strains with a degron‐regulated Dcp2 protein, when protein degradation was not induced (dcp2‐deg) or was induced by addition of auxin (dcp2‐deg + auxin) and incubation for 1 h. Values represent average and standard deviation for at least three independent experiments.

Figure 6. Upf2 and Upf3 function as a heterodimer in the formation of Upf1‐23

1. A, B

   Comparison between average enrichment values for Upf1‐decapping and Upf1‐23 components, Lsm proteins and CK subunits in Upf1‐TAP‐purified samples in the presence (grey bars) or the absence (blue bars) of UPF2 (A) or UPF3 (B). Error bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1‐TAP, 3 for the upf2Δ and upf3Δ conditions.

2. C, D

   Evaluation of the effects of UPF2 deletion on Upf3‐TAP levels (C) and of UPF3 deletion on Upf2‐TAP levels (D) in total extracts, in comparison with a loading control (G6PDH), was done by immunoblot.

3. E

   Comparison between average enrichment values for Upf1‐decapping and Upf1‐23 components, Lsm proteins and CK subunits in Upf2‐TAP‐purified samples in the presence (grey bars, six experiments) or the absence (blue bars, two experiments) of UPF1. Error bars represent SD. The levels of expression of Upf2‐TAP and Upf3‐TAP proteins have been verified in this condition (Appendix Fig S2).

Source data are available online for this figure.

Figure 7. Association of Nmd4 with NMD substrates depends on Upf1‐23

1. A

   Unspliced RPL28, an NMD substrate, was enriched in Nmd4‐TAP purification in comparison with a control untagged strain, as measured by reverse transcription followed by quantitative PCR. Bars correspond to the mean of pre‐RPL28 enrichment for three independent experiments, as compared with RIM1, a non‐NMD mRNA. Error bars correspond to SD. The indicated P‐value was computed using the Welch t‐test, single‐sided comparison.

2. B–D

   Distribution of Nmd4‐TAP, Nog1 (marker of free 60S subunits) and Rps8 (marker of 40S, 80S and polysome fractions) in wild‐type (B), upf1∆ (C) and upf2∆ (D) strains, tested by immunoblot. Fractions 1, 2, 3 are light fractions; fractions 4, 5 and 6 correspond, respectively, to ribosomal 40S, 60S and 80S positions; fractions 7–12 correspond to polysomes. The per cent of total signal for Nmd4‐TAP in three independent replicates along with standard deviation of the values are indicated for each fraction.

3. E

   Quantified relative changes in Nmd4‐TAP average levels in light and monosome/polysome fractions in upf1∆ and upf2∆ strains (100% correspond to levels in the wild‐type strain). Indicated P‐values correspond to the Welch t‐test, single‐sided comparison of four replicate experiments (individual values are indicated as dots). Error bars correspond to SD.

4. F

   Summary of the Upf1 domains interaction with Upf1‐23 and Upf1‐decapping components, based on the data presented in this work.

5. G

   Proposed Upf1‐23/Upf1‐decapping sequence of events for yeast NMD.

Source data are available online for this figure.