ATPase-deficient hUpf1 accumulates on the 3’ NMD intermediate

How does the Upf1 ATPase stimulate degradation of the 3’ NMD fragment by Xrn1? One possibility is that the Upf1 ATPase triggers removal of protein from the 3’ NMD mRNP intermediate, thereby allowing access for Xrn1. If so, it is predicted that wild-type hUpf1 should cycle off the 3’ NMD intermediate, whereas ATPase-deficient hUpf1 should fail to do so. This was tested using hUpf1 immunoprecipitation (IP) followed by Northern blotting for associated NMD substrate mRNA under strong Xrn1 knock-down conditions. As seen in Figure 2A, both wild-type and mutant hUpf1 proteins associate with full-length β-39 NMD substrate produced by a short transcriptional pulse (lanes 6–8). However, the association of the accumulating 3’ β-39 fragment with ATPase-deficient hUpf1 is strongly enhanced (4.1-fold relative to full-length β-39) as compared with wild-type hUpf1 (Figure 2A; compare lanes 7 and 8 to lane 6; band marked by arrow). These interactions occur in the cell and do not form after cell lysis, as β-39 mRNA does not co-purify with wild-type or mutant hUpf1 when expressed in separate cells and combined during cell lysis (Figure 2B; compare lanes 6 to 5 and 12 to 11), and the mRNA substrate does not co-purify with the antibody in the absence of exogenous hUpf1 (Figure 2A, lane 5; Figure 2B, lanes 4, 10). These observations are consistent with the idea that ATPase activity is not critical for recruitment of hUpf1 to the NMD substrate, but is required for the release of hUpf1 from the 3’ fragment that forms after initiation of mRNA decay.

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Figure 2

The 3’ NMD endonucleolytic cleavage fragment is stuck with ATPase-deficient Upf1 and is resistant to 5'-to-3' exonucleolytic decay in vitro

(A) Northern blot for β-39 mRNA from pellet (lanes 5–8) or 5% of total extract (lanes 1–4) fractions from anti-myc IP assays from cells transiently expressing myc-tagged hUpf1 proteins indicated on the top or no exogenous protein (none). Cells were treated with Xrn1 #2 siRNA to promote the accumulation of the β-39 mRNA 3’ fragment.

(B) Same as (A), but β-39 mRNA was expressed either in the same cells as wild-type (WT) or DEAA mutant (DE) hUpf1 (lanes 2, 5, 8, 11), or in different cells and mixed prior to extract preparation (lanes 3, 6, 9, 12). Lanes 1–3, 7–9: 5% of total extracts; Lanes 4–6, 10–12: IP pellets. Lanes 1, 4, 7 and 10 are from cells not expressing Myc-hUpf1. All cells were treated with Xrn1 #2 siRNA to promote the accumulation of the β-39 mRNA 3’ fragment.

(C) Northern blots showing in vitro Terminator-mediated decay of β-39 3' mRNA fragment from extracts (left panels) or total RNA (right panels) from HeLa Tet-off cells depleted of endogenous hUpf1 using an siRNA and expressing exogenous siRNA-resistant wild-type hUpf1 (hUpf1^R) or hUpf1 ATPase mutants. An siRNA targeting Xrn1 (Xrn1 #2) was included in all experiments. Time points above each lane represent the time of Terminator incubation. Bottom panels: incubation in the absence of Terminator.

(D) Quantification for each of the experiments in (C). Percent and standard deviation are calculated from three experiments.

See also Figure S2.

The 3’ NMD mRNP fragment generated in the presence of ATPase-deficient Upf1 is resistant to 5’-to-3’ exonucleolytic decay in vitro

If the 3' NMD intermediate that forms in the presence of hUpf1 ATPase mutants is resistant to Xrn1 due to a failure in mRNP disassembly, it should become sensitive to 5'-to-3' exonucleolytic decay if protein is removed from the mRNP. To test this in vitro, β-39 mRNA was expressed along with exogenous wild-type or ATPase-deficient hUpf1 proteins in HeLa Tet-off cells depleted for endogenous Xrn1 and hUpf1. Cells were subsequently permeabilized and the resulting cell extracts were incubated with the Terminator enzyme, a commercially available 5’-to-3’ exonuclease. As seen in Figure 2C (left panels), while the 3’ NMD mRNA fragment that accumulates due to Xrn1 knock-down in the presence of wild-type hUpf1 is degraded efficiently (t_1/2≈18 mins), a large fraction (60–70%) of the same RNA produced in cells expressing ATPase-deficient Upf1 proteins is highly resistant to 5'-to-3' exonucleolytic decay (quantified in Figure 2D, top panel). By contrast, when mRNPs were disrupted and protein removed from the cell extracts by phenol extraction prior to incubation with the nuclease, the 3’ NMD fragment is degraded efficiently regardless of the ability of Upf1 to hydrolyze ATP (Figure 2C, right panels; quantified in Figure 2D, bottom panel). As expected, full-length β-39 mRNA is resistant to the Terminator enzyme, which is specific for 5’ monophosphate-containing RNA and thus does not target capped RNA (Figure 2C; upper bands), and the 3’ fragment does not degrade in the absence of Terminator (bottom panels). When the endonuclease RNase A was used in place of Terminator, all RNAs rapidly degrade (Figure S2). Thus, when Upf1 fails to hydrolyze ATP, the 3' NMD fragment generated by Smg6-mediated endonucleolytic cleavage becomes trapped in an mRNP that includes hUpf1 and is resistant to exonucleolytic decay from the 5’ end.

The 3’ NMD intermediate accumulates in PBs in the presence of ATPase-deficient Upf1

A number of studies have demonstrated that cytoplasmic mRNPs that accumulate in association with 5’-to-3’ mRNA decay complexes concentrate in PBs (Eulalio et al., 2007; Franks and Lykke-Andersen, 2008; Parker and Sheth, 2007). Thus, if hUpf1 ATPase activity is critical for NMD mRNP disassembly during mRNA decay, it is predicted that the NMD intermediate should accumulate in PBs when hUpf1 fails to hydrolyze ATP. Indeed, as seen in the fluorescence in situ hybridization (FISH) assays in Figure 3A, both β-39 (panels 2 and 3) and GPx1-46 (panels 5 and 6) mRNAs accumulate strongly in PBs in the presence of ATPase-deficient hUpf1, but are rarely detected when wild-type hUpf1 is expressed (panels 1 and 4). This is consistent with previous observations in yeast (Sheth and Parker, 2006). By contrast, wild-type β-globin mRNA accumulated only at very low levels in PBs upon mutant hUpf1 expression (Figure S3).

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Figure 3

The 3’ NMD intermediate accumulates in PBs in the presence of ATPase-deficient hUpf1

(A), (B) and (C) FISH assays showing localization of β-39, β-ARE and GPx1-46 mRNAs in HeLa cells in which endogenous hUpf1 was replaced with exogenous hUpf1, hUpf1 DEAA or hUpf1 K498A as indicated above the panels. Individual Texas-Red-labeled 50-nt probes targeting various regions of β-globin and GPx-1 mRNAs were used in (B) and (C) as indicated below images, whereas equimolar amounts of all probes were used in the experiments in (A). GFP-hDcp1a was used as a PB marker. Merged images are displayed (RNA:red, GFP-hDcp1a:green) while selected enlarged regions are shown unmerged below. Percentage of cells displaying mRNA signal in PBs is shown in the bottom right corner of images (with the number of cells counted from at least three experiments in parentheses), and graphed for individual probes against β-39 or β-ARE mRNA below cell images in (B). Note: plasmids that express β-39, β-ARE and GPx1-46 mRNAs also express GFP; thus some nuclear staining can be observed in the green channel.

See also Figure S3.

We next used individual probes hybridizing to different regions along the β-globin mRNA to ask which part of the NMD substrate accumulates in PBs. Remarkably, while the region 3’ of the PTC of β-39 mRNA was readily detectable in PBs in the presence of ATPase-deficient hUpf1, the 5’ end remained completely undetectable in PBs (Figure 3B, compare panels 4 and 5 with panels 1 and 2; quantifications below), despite the fact that the full-length mRNA under these conditions is 6- to 10-fold more abundant than the 3’ fragment (Figures 1A and 1B). A probe that hybridizes across the mapped Smg6 endonucleolytic cleavage sites (Eberle et al., 2009) modestly detects the mRNA in PBs (panel 3). The observed differences in PB detection are not due to different efficiencies of the FISH probes, since in contrast to the β-39 mRNA, each FISH probe equally detected in PBs a β-globin mRNA targeted for the ARE-mRNA decay pathway (β-ARE) (compare panels 6–10 with panels 1–5; quantifications below). The observed localization pattern is not unique to the β-39 mRNA, as in the presence of ATPase-deficient hUpf1 the 3’ end of an unrelated NMD substrate, GPx1-46, could also be observed in PBs in contrast to its 5’ end (Figure 3C). Thus the 3' NMD mRNA decay intermediate that accumulates when Upf1 fails to hydrolyze ATP forms an mRNP that concentrates in PBs.

Multiple NMD factors accumulate in PBs in the absence of Upf1 ATPase activity

What are the protein components of the accumulating 3’ NMD mRNP intermediate? Based on the observations above, such proteins are predicted, 1) to accumulate in PBs in the presence of ATPase-deficient Upf1, 2) to co-purify more strongly with ATPase-deficient Upf1 than with wild-type Upf1 in co-immunoprecipitation (co-IP) assays, and 3) to co-purify the NMD mRNA 3’ fragment when immunoprecipitated. We tested these predictions for multiple NMD factors. Consistent with hUpf1 being part of the 3’ NMD mRNP and with previous observations in yeast and human cells (Sheth and Parker, 2006; Cheng et al., 2007; Cho et al., 2009; Stalder and Muhlemann, 2009), indirect immunofluorescence assays revealed that ATP binding- and ATPase-deficient mutant hUpf1 proteins, but not wild-type hUpf1, accumulate strongly in PBs (Figure 4, compare panels 4, 7, 10, 13 to panel 1). This is consistent with the observation that ATPase activity is required for the release of hUpf1 from the degrading NMD mRNP (Figure 2A).

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Figure 4

Mutant hUpf1 proteins deficient in ATP binding or ATP hydrolysis accumulate in PBs

Indirect immunofluorescence assays showing localization of myc-tagged wild-type hUpf1, ATPase mutant (DEAA), or ATP-binding mutants (K498A, G494R, G496E) hUpf1 proteins transiently expressed in HeLa cells (left panels). Human IC-6 serum, which detects the decapping factor Hedls and the nuclear envelope component Lamin, was used as a PB marker (middle panels). Merged images (hUpf1: green; IC-6: red) are shown in the right panels. Enlarged images of the indicated boxed areas are shown in the upper left corner for each image.

What about other NMD factors? Remarkably, exogenously expressed ATPase-deficient hUpf1 (Figure 5A), but not wild-type hUpf1 (Figure 5B), induces strong accumulation of endogenous Smg5, Smg6 and Smg7 in PBs (panels 4, 7 and 10; transfected cells identified by the co-expression of NLS-DsRed are marked by arrowheads) but has no observable effect on the localization of an unrelated RNA binding protein, HuR (panel 28). Smg1, hUpf2 and the EJC components Y14 and eIF4A3 more modestly accumulate in PBs (panels 13, 16, 19, and 22), whereas hUpf3a and hUpf3b were only rarely observed in PBs (our unpublished observations). None of the NMD factors localized strongly in PBs in untransfected cells (Figures 5A and B; cells not indicated by arrowheads), which in all cases looked similar to those expressing exogenous wild-type hUpf1 (Figure 5B). Similarly to NMD factors, Xrn1 consistently showed enhanced accumulation in PBs in cells expressing ATPase-deficient hUpf1 (Figure 5A, panel 25; cell marked by arrowhead) as compared with cells expressing exogenous wild-type hUpf1 (Figure 5B, panel 25) or no exogenous hUpf1 (Figures 5A and B, panel 25; unmarked cells). Thus, multiple NMD factors and Xrn1 co-accumulate with NMD intermediates in PBs in the presence of ATPase-deficient hUpf1 (Figure 5).

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Figure 5

Multiple NMD factors accumulate in PBs in the presence of ATPase-deficient hUpf1

(A) and (B) Indirect immunofluorescence assays showing localization in HeLa cells of endogenous NMD factors as indicated on the left, or a protein not involved in NMD, HuR, in the presence of exogenously expressed (A) hUpf1 DEAA, or (B) wild-type hUpf1. Middle panels show human IC-6 serum as a PB marker and DsRed with a nuclear localization signal to mark transfected cells (indicated by white arrowheads). Merged images (NMD factor: green; IC-6/NLS-DsRed: red) are shown in right panels. An enlarged cell section representing the boxed area of each image is shown in the upper left corner. The average enrichment of the protein factor in PBs over the general cytoplasm was quantified in transfected cells and given with standard deviation in each of the panels on the left.

ATPase-deficient hUpf1 shows enhanced co-purification with multiple NMD factors

We next tested the prediction that proteins that require Upf1 ATPase activity for release from the NMD mRNP should co-purify more strongly with ATPase-deficient hUpf1 mutant proteins than with wild-type hUpf1 in co-IP assays. In striking correlation with the immunofluorescence assays above, the co-IP assays in Figure 6A (lanes 1–8), in which cell extracts where not treated with RNase, show strong enrichment of endogenous Smg5 and Smg7, and exogenous Smg6, in hUpf1 ATPase mutant protein complexes as compared with wild-type hUpf1 complexes (compare lanes 3 and 4 with lane 2; see also Figure S4). Other NMD factors, hUpf2, hUpf3b and eIF4A3 show modestly enhanced accumulation with hUpf1 ATPase mutant complexes (Figure 6A), which correlates well with their moderate accumulation in PBs under the same conditions (Figure 5A; our anti-hSmg6 and anti-hSmg1 antibodies failed to detect the endogenous proteins on Western blots). These observations are consistent with previous observations of enhanced association of ATP binding-deficient hUpf1 with Smg7, hUpf3a and hUpf2 (Kashima et al., 2006). β-actin served as a negative control and did not co-purify with wild-type or mutant hUpf1 proteins (Figure 6A, bottom panel), and none of the endogenous NMD factors non-specifically co-purified with the IP resin (lane 1). When the same assays were repeated in the presence of RNase, Smg5 and Smg7 were the only NMD factors that remained enriched in the mutant hUpf1 complexes, suggesting the accumulation of an RNA-independent interaction between these factors when hUpf1 fails to hydrolyze ATP (Figure 6A, lanes 9–16). In addition to NMD factors, both Xrn1 and PABPC1 showed enhanced association with ATPase-deficient hUpf1 proteins over wild-type hUpf1, although this was more evident in the presence than in the absence of RNase-treatment (compare lanes 11,12 to 10 and 3,4 to 2). Unlike Xrn1 and NMD factors, PABPC1 was not observed to concentrate in PBs upon ATPase-deficient hUpf1 expression (our unpublished observations), perhaps due to the high cytoplasmic abundance of PABPC1 overwhelming detection in PBs. Taken together, these observations are consistent with the idea that the hUpf1 ATPase stimulates disassembly of the NMD mRNP. However, some NMD factors show stronger accumulation than others in the trapped mRNP complexes (Figures 5 and ​and66).

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Figure 6

Multiple NMD factors accumulate in complex with ATPase-deficient hUpf1 and the NMD substrate 3’ fragment

(A) Western blots for the proteins indicated on the left from pellet (left panels) or 2% of total extract (right panels) fractions from anti-myc IP assays from HEK 293T cells transiently expressing proteins shown on the top, or no exogenous protein (none). Cell extracts in lanes 9–16 were treated with RNase A prior to IP.

(B) Northern blots for β-39 mRNA isolated from pellets (IP; top panels) or 5% total extract (Total; bottom panels) fractions from immunoprecipitation reactions for tagged exogenous, or in the case of hUpf2, endogenous, NMD, EJC or PABPC1 factors, as shown on the top, in the presence of co-expressed wild-type (WT), or ATPase-deficient (DEAA) hUpf1. Endogenous hUpf1 and Xrn1 were knocked down using siRNAs. (−) indicates a reaction using anti-HA beads in the absence of HA-tagged protein. Anti-FLAG and anti-Myc beads looked similar (not shown). Below each panel is shown the calculated enrichment of the 3’ fragment relative to full-length β-39 mRNA in IP pellets in the presence of mutant hUpf1 (DEAA) over that in the presence of wild-type hUpf1. Representative of three independent experiments.

See also Figure S4.

Is mRNP disassembly a regulated step in NMD?

Taken together, our observations uncover a previously unappreciated ATP-dependent mRNP disassembly step in mRNP turnover. A key question is what controls the timing of mRNP disassembly in NMD, as slow disassembly would cause accumulation of decay intermediates whereas rapid disassembly could potentially release the NMD mRNP even before it initiates decay. The ATPase activity of human Upf1 is stimulated by the Upf2-Upf3 complex (Chamieh et al., 2008), and the yeast Upf1 ATPase is repressed by translation release factors eRF3 and eRF1 (Czaplinski et al., 1998). Thus, a transition in the NMD mRNP in which Upf1 is released from eRFs and associates with Upf2-Upf3 may precede activation of the Upf1 ATPase and subsequent mRNP disassembly. Consistent with this, ATP binding-deficient Upf1 has been observed to co-purify less efficiently than wild-type Upf1 with eRF1 and eRF3 (Czaplinski et al., 1998; Kashima et al., 2006; Isken et al., 2008), suggesting that it becomes trapped in a complex lacking eRFs. Moreover, analyses of NMD complexes stalled by NMD factor mutation or depletion have indicated a transition in the human NMD mRNP from a complex between Upf1, Smg1 and eRFs (called SURF) to a complex of NMD factors lacking eRFs (called DECID) (Kashima et al., 2006). In addition, the phosphorylation and dephosphorylation of metazoan Upf1 seems to be coordinated with the Upf1 ATPase, as ATPase-deficient Upf1 accumulates in a hyperphosphorylated form (Isken et al., 2008; Kashima et al., 2006; Page et al., 1999), which has been reported to prevent translation re-initiation on the NMD mRNP (Isken et al., 2008).

Why would mRNP disassembly be under such tight control during NMD? This could possibly ensure that NMD factors are released only after mRNA decay factors have already been recruited and/or mRNA decay initiated. This also raises the possibility that ATPase-mediated mRNP disassembly could serve as a previously proposed proof-reading step in the NMD pathway (Sheth and Parker, 2006), in which rapid hydrolysis of ATP by Upf1 would allow the release of the NMD machinery from the mRNA even before initiation of mRNA decay, thus allowing mRNAs wrongly targeted for NMD to be released prior to decay (Figure 7). Several lines of evidence suggest that the composition of the mRNP downstream of the translation termination event controls NMD (Amrani et al., 2006; Muhlemann, 2008; Rebbapragada and Lykke-Andersen, 2009). A key question for future studies is whether the downstream mRNP controls NMD not just by NMD factor recruitment as has been generally assumed, but also in part by regulating the Upf1 ATPase.

Indirect Immunofluorescence and Fluorescence In Situ Hybridization (FISH) Assays

Human HeLa cells transiently expressing wild-type or mutant myc-tagged hUpf1 proteins were fixed with formaldehyde and permeabilized with Triton-X100 (Figures 4 and ​and5)5) or ethanol (Figure 3). For indirect immunofluorescence assays, cells were incubated with antibodies against Myc-tag (Figure 4) or against endogenous NMD factors, Xrn1 or HuR (Figure 5) as well as with human IC-6 serum, which recognizes endogenous Hedls (P-body marker) and Lamin, followed by fluorescently labeled secondary antibodies (anti-mouse or -rabbit: Alexa 488; anti-human: Texas Red). Cells in Figure 5 express nuclear DsRed to mark transfected cells. For FISH assays, cells were hybridized with a mixture of (Figures 3A, C), or individual (Figure 3B), TexasRed-5’-labeled 50-nucleotide NMD substrate mRNA antisense DNA probes. Cells for FISH assays express GFP-tagged hDcp1a to mark P-bodies (see Supplemental Experimental Procedures for details).

We thank Drs. Tom Blumenthal (University of Colorado), Melissa Moore (University of Massachusetts Medical Center) and Sebastien Durand (UCSD) for comments on the manuscript. Alex Choe and Claire Egan are thanked for technical support and Joachim Weischenfeldt for production of the antigen for anti-Smg1 antibodies. Drs. Marv Fritzler, Ed Chan and Donald Bloch are thanked for human IC-6 serum. Dr. Oliver Mühlemann is thanked for the HA-Smg6 construct. Work on P-bodies in our laboratory is supported by funding from grant R01 GM077243 from the National Institutes of Health to J.L.-A. T.M.F. has been supported by National Institutes of Health NRSA Institutional Training Grant no. GM-07135 from the National Institute of General Medical Sciences.