Upf1p, Upf2p, and Upf3p Can Localize to P-Bodies
Since Upf1p, Upf2p, and Upf3p are required for NMD, the presence of these proteins in P-bodies would argue for the occurrence of NMD in P-bodies. To address this issue, a C-terminal GFP fusion of each Upf protein was constructed and the cellular localization of the chimeras determined in wild-type cells. Northern analysis of the CYH2 pre-mRNA, which is a substrate for NMD (He et al., 1993
), indicated that the tagged Upf1p, Upf2p, and Upf3p are fully functional (data not shown). Consistent with prior work (Atkin et al., 1995
; Shirley et al., 1998
), GFP-tagged Upf1p, Upf2p, and Upf3p were uniformly distributed in the cytoplasm (, top panel). However, if Upf proteins target mRNA to P-bodies but are released rapidly after the degradation of the mRNA, they might only accumulate in P-bodies if degradation of the mRNA within P-bodies were blocked.
Upf Proteins Are Localized to P-Bodies
To test this model, we localized the Upf proteins in strains blocked at the catalytic steps of decay. In dcp1Δ, dcp2Δ, and xrn1Δ strains, Upf1p, Upf2p, and Upf3p were present in cytoplasmic foci (, bottom panel and data not shown), which in the dcp1Δ strain colocalized with Dcp2p-RFP (). Quantification of the images (see Experimental Procedures) indicated an increase in both the number and area of Upf-GFP signal within P-bodies (). These foci, and all other microscopic data presented in this manuscript, unless explicitly stated, were seen in greater than 80% of cells examined. This observation suggests that the Upf proteins cycle through P-bodies and accumulate in P-bodies when decapping is blocked.
Since Dcp1p is involved in the decay of both normal and nonsense-containing mRNAs (Beelman et al., 1996
), the accumulation of Upf proteins in P-bodies in a dcp1Δ strain could be due to the accumulation of normal and/or nonsense-containing mRNAs in these foci. To determine if the accumulation of Upf proteins in P-bodies was specific to NMD, we examined the distribution of Upf1p-GFP in a lsm1Δ strain, which is only defective in the decapping of only normal mRNAs (Boeck et al., 1998
). In the lsm1Δ strain, Upf1p-GFP is not concentrated in P-bodies (, middle panel) as compared to a dcp1Δ strain (, bottom panel) or a dcp2Δ strain (, right panel, and 1E). Similar results were also obtained for Upf2p-GFP and Upf3p-GFP (data not shown). In contrast, Dhh1p, an activator of decapping involved in the 5′ to 3′ decay of normal mRNA (Coller et al., 2001
), accumulated in P-bodies in lsm1Δ, dcp1Δ, and dcp2Δ strains (, middle panel and left panel and 1G and data not shown). This result indicates that the presence of Upf proteins in P-bodies is due to a defect in NMD and implies that NMD targets PTC-containing mRNAs to P-bodies.
A Reporter mRNA Harboring a Nonsense Mutation Localizes to P-Bodies
To directly test if NMD targets mRNAs to P-bodies, we examined the subcellular distribution of PGK1 mRNAs with early (codon 22) or late (codon 225) nonsense codons. We examined reporter mRNAs with early and late nonsense codons since they differ in their rates of decapping, have different rate-limiting steps in decay, and therefore may respond differently in the NMD process (Cao and Parker, 2003
). These reporter mRNAs contained 16 U1A binding sites in the 3′ UTR of the mRNA, which did not prevent NMD (Figure S1), and a U1A-GFP fusion protein was coexpressed, which allowed visualization of transcript localization (Bertrand et al., 1998
; Brodsky and Silver, 2000
; Teixeira et al., 2005
). When expressed by itself, the U1A-GFP fusion protein showed no accumulation in P-bodies (, top panel, and ). As compared to the wild-type (wt) PGK1 mRNA, both the C22 and C225 mRNAs showed an increase in the number of P-bodies and their overall area, as detected by the tethered U1A-GFP (, right panel compare panel v (wt) to panel ix (C22) and panel xiii (C225) and ). Furthermore, these mRNA-containing foci colocalized with RFP-tagged Dcp2p (Figure S2). These results show that PTC-containing mRNAs accumulate in P-bodies.
Nonsense-Containing Full-Length Reporter mRNA Localizes to P-Bodies
To determine if the accumulation of the C22 and C225 mRNAs in P-bodies was due to NMD, we examined how this localization was affected in upf1Δ, upf2Δ, and upf3Δ strains. In upf1Δ strain, the accumulation of both the C22 and C225 reporter mRNAs in P-bodies was reduced both in the number of P-bodies observed and their overall area (; ). The continued accumulation of C22 mRNA in P-bodies in the upf1Δ strain is likely to be due to the short ORF on this transcript, and the inverse relationship seen between an mRNA being associated with ribosomes or accumulating in P-bodies (Brengues et al., 2005
; Teixeira et al., 2005
). It should be noted that the decrease in P-body accumulation in the upf1Δ strain was observed despite an increase in the levels of the C22 and C225 mRNAs in the upf1Δ strain due to an inhibition of NMD (Figure S1). This observation argues that Upf1p is required for the targeting of NMD substrates to P-bodies.
The C22 and C225 mRNAs behaved slightly differently in upf2Δ and upf3Δ strains. The C22 mRNA showed an approximately 2-fold increase in P-bodies both in number and area in upf2Δ and upf3Δ strains as compared to the upf1Δ strain (). The C225 mRNA also accumulated more detectable P-bodies in upf2Δ and upf3Δ strains as compared to the upf1Δ strain, although the overall area of these P-bodies was not significantly different (). These results suggest that Upf1p is the critical protein for targeting mRNAs to P-bodies whereas Upf2p and Upf3p function, at least in part, after P-body targeting of the mRNA (see below). However, the reduction of the C225 mRNA in P-bodies in upf2Δ and upf3Δ strains as compared to wt strain suggests that Upf2p and Upf3p might also affect the efficiency with which the C225 mRNA is targeted to P-bodies (see Discussion).
Despite the differences in subcellular mRNA location, all three Upf proteins are required for the rapid decay of the C22 (early PTC) and C142 (intermediate PTC) transcripts (Figure S3 and data not shown). This suggests that localization of the PTC-containing mRNA to P-bodies by Upf1p is not sufficient for its degradation and that Upf2p and Upf3p are required to trigger decay of the mRNA.
Upf1p Acts upstream of Upf2p and Upf3p
The above observations suggest that a substep in NMD is the targeting of the mRNA to a specific mRNP complex, which can accumulate, and be detected, as P-bodies. This accumulation of mRNAs and proteins in P-bodies then becomes an assay for a substep in the NMD process. Thus, we examined whether each Upf protein acts upstream or downstream of P-body formation by using Dcp2p-GFP to monitor P-body formation in upf mutants. Both the number and overall area of Dcp2p-GFP in P-bodies increased in upf2Δ, upf3Δ, and upf2Δupf3Δ strains (, compare i to iii, iv, vii, and viii). In contrast, Dcp2p-GFP in the upf1Δ strain was distributed similarly to the wild-type strain (, compare i and ii, and viii). Moreover, GFP-tagged Dcp2p did not show increased accumulation in P-bodies in the upf1Δupf2Δ or upf1Δupf3Δ strain (), indicating that the accumulation of P-bodies in upf2Δ and up3Δ strains is dependent on Upf1p. The changes in the P-bodies are not due to differences in Dcp2p-GFP abundance since the level of Dcp2p-GFP does not significantly differ in these strains (Figure S4). These observations argue that Upf1p functions upstream of Upf2p and Upf3p.
To identify other proteins that accumulate in P-bodies in upf2Δ and upf3Δ strains, we examined the localization of Dcp1p-GFP, Xrn1-GFP, Dhh1p-GFP, Pat1p-GFP, and Lsm1p-GFP in different upf mutants. As assessed by both average P-body number and area, Dcp1p, Xrn1p, Dhh1p, Pat1p, and Lsm1p accumulated in P-bodies in upf2Δ and upf3Δ strains ( and data not shown). This observation suggests two possibilities. First, normal mRNAs in the process of decay might accumulate in P-bodies in upf2Δ and upf3Δ strains. However, this possibility is unlikely since normal mRNAs do not accumulate in P-bodies in upf2Δ and upf3Δ strains (). More plausibly, when Upf1p targets an mRNA to assemble a P-body complex it may recruit the decapping enzyme as part of a larger complex consisting of the decapping enzymes (Dcp1p/Dcp2p), activators of decapping (Dhh1p, Pat1p, and Lsm1–7p), and the 5′ to 3′ exonuclease Xrn1p.
Interdependence of Upf Proteins
Examining the location of each of the Upf proteins in strains lacking one of the other two Upf proteins provided three observations. First, Upf1p-GFP accumulated in P-bodies in upf2Δ or upf3Δ and upf2Δupf3Δ strains ( and data not shown). This further supports the model that Upf1p functions upstream, and independently, of Upf2p and Upf3p. This result also implies that Upf2p and Upf3p are required for degradation of the mRNA after P-body targeting. A second observation was that Upf2p and Upf3p do not accumulate in P-bodies in an upf1Δ strain () consistent with Upf1p acting upstream of Upf2p and Upf3p. Third, in contrast to Upf1p, neither Upf2p nor Upf3p accumulated in foci in upf3Δ or upf2Δ strains, respectively (). This suggests that the stable accumulation of Upf2p and Upf3p in P-bodies is interdependent.
These results argue that Upf1p is required for targeting of PTC-containing mRNAs to P-bodies. In contrast, in strains lacking Upf2p and Upf3p, mRNAs and associated proteins accumulate in P-bodies, presumably due to the action of Upf1p, but are not degraded. This is also supported by the observation that the localization of a reporter mRNA harboring an early PTC to P-bodies is decreased in upf1Δ, whereas its levels increase in upf2Δ and upf3Δ strains ().
Upf1p Has a Central Role in Targeting mRNAs to P-Bodies
Upf1p is a 5′ to 3′ ATP-dependent RNA helicase and its ATPase activity is essential for NMD in an unknown manner (Weng et al., 1996
). One possibility is that the ATPase activity of Upf1p may be required for the initial targeting of mRNAs to P-bodies. This model predicts that a strain expressing an ATPase-defective allele of Upf1p would not be able to target mRNAs to P-bodies and therefore P-bodies would decrease in size. Alternatively, the ATPase domain could be required for degradation of the mRNA substrate, after targeting to a P-body. In this case, a strain expressing an ATPase-defective allele of Upf1p would show an increase in P-bodies.
To test these predictions, we expressed wt Upf1p and DE572AA, an ATPase-defective upf1
allele (Weng et al., 1996
) from a low-copy plasmid in an upf1Δ strain and examined P-body number and area. We observed that Dcp2p-GFP accumulated in P-bodies in cells expressing the DE572AA upf1
allele () but not in strains containing either wt Upf1p or empty vector (). This suggests that the ATP hydrolysis activity of Upf1p is not required for P-body targeting and instead may promote a rearrangement of the mRNP within P-bodies that can trigger mRNA degradation.
Analysis of the DE572AA upf1 Allele
Additional evidence for the role of the ATP hydrolysis activity of Upf1p comes from overexpression of the DE572AA upf1 allele from a two-micron plasmid in wild-type strains expressing GFP-tagged Upf1p, Upf2p, Upf3p, Dhh1p, Pat1p, or Lsm1p. GFP-tagged Upf1p, Dhh1p, Pat1p, and Lsm1p localized to P-bodies in strains overexpressing the DE572AA upf1 allele (). Overexpressing wt Upf1p, or presence of empty vector, had no effect on the localization of all the proteins tested except for Upf1p where overexpression of wt Upf1p led to a slight increase in P-bodies (). Moreover, the accumulation of P-bodies in strains overexpressing the DE572AA upf1 allele is independent of Upf2p and Upf3p, as shown by the accumulation of Dcp2p-GFP in upf2Δ and upf3Δ strains (). In contrast to Upf1p, GFP-tagged Upf2p and Upf3p did not localize to P-bodies in strains overexpressing either wt Upf1p or the DE572AA upf1 allele (). This observation indicates that either the DE572AA upf1 allele may have lost its ability to recruit Upf2p or Upf3p or that Upf2p and Upf3p may associate with the mRNP after the ATP hydrolysis step.
Upf1p Can Target Normal mRNAs to P-Bodies
The accumulation of proteins in P-bodies in the DE572AA allele of Upf1p argues that some mRNAs are accumulating in P-bodies in this strain. Thus, we examined the localization of both normal and nonsense mRNAs in an upf1Δ strain expressing the DE572AA upf1 allele. The P-body accumulation of the PTC (C22)-containing PGK1 reporter mRNA slightly increased in the strain expressing the DE572AA upf1 allele as compared to a strain expressing wt Upf1p (, bottom panel compare left to right, and 5B, bottom panel). These results argue that ATP hydrolysis by Upf1p is not required for localization of nonsense mRNAs, or associated proteins, to a P-body.
ATPase Activity of Upf1p Is Not Required for Targeting of mRNAs to P-Bodies
Surprisingly, the expression of the DE572AA upf1
allele also led to clear accumulation of wt mRNAs in P-bodies as assessed both by the number of P-bodies containing this mRNA and their overall area (, top panel compare left to right, and 5B, top panel). This observation implies that Upf1p can also target normal mRNAs to P-bodies and that they accumulate in P-bodies either because ATP hydrolysis is required for their degradation or for their release back into translation. Transcriptional pulse-chase experiments, which allow measurement of the rates of both deadenylation and decapping of the mRNA (Cao and Parker, 2003
), indicated that deadenylation, decapping, and overall decay rates of the wt PGK1 mRNA were not affected by the DE572AA upf1
allele (). Moreover, we did not observe even a small pool of mRNAs which might be stuck in a nonproductive intermediate in the DE572AA allele and thereby be responsible for the increased P-bodies. This suggests that the ATP hydrolysis activity of Upf1p is likely to be required to release a pool of normal mRNAs that can be targeted to P-bodies by Upf1p back into the translating pool.
The observation that Upf1p leads to the accumulation of normal mRNAs in P-bodies suggests that Upf1p can repress the translation of normal mRNAs. This predicts that overexpression of Upf1p might generally affect translation. To test this possibility, we overexpressed either wt Upf1p or the DE572AA upf1 allele from the galactose promoter in wild-type strain and examined P-body formation and polysome profiles 2 hr after induction with galactose. Compared to vector alone (), overexpression of the DE572AA upf1 allele reduced polysomes (, compare the red polysome trace [sucrose] to the gray [galactose] polysome trace) and showed a clear accumulation of P-bodies (, compare ii to iii). In addition to a reduction in polysomes and an increase in P-body size, overexpression of the DE572AA upf1 allele also inhibited growth (, compare iv to v) whereas overexpression of wt Upf1 caused a minor, but clear, decrease in growth rate (, compare iv to v). These results are consistent with Upf1p repressing the translation of a pool of ‘‘normal’’ mRNAs and targeting those transcripts to P-bodies. Moreover, because the DE572AA allele is more severe than overexpression of the wt Upf1p, it implies that the DE572AA is inhibiting the recycling of ‘‘normal’’ mRNAs back into translation (see Discussion).
Overexpression of DE572AA upf1 Allele Causes Translational Repression, an Increase in P-Bodies, and Inhibition of Growth