A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay
2007; Springer Nature; Volume: 26; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7601588
ISSN1460-2075
AutoresIsabelle Behm‐Ansmant, David Gatfield, Jan Rehwinkel, Valérie Hilgers, Elisa Izaurralde,
Tópico(s)RNA regulation and disease
ResumoArticle22 February 2007free access A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay Isabelle Behm-Ansmant Isabelle Behm-Ansmant Max-Planck-Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author David Gatfield David Gatfield EMBL, Heidelberg, GermanyPresent address: University of Geneva, Quai Ernest-Ansermet, 1211-Geneva, Switzerland Search for more papers by this author Jan Rehwinkel Jan Rehwinkel EMBL, Heidelberg, Germany Search for more papers by this author Valérie Hilgers Valérie Hilgers EMBL, Heidelberg, Germany Search for more papers by this author Elisa Izaurralde Corresponding Author Elisa Izaurralde Max-Planck-Institute for Developmental Biology, Tübingen, Germany EMBL, Heidelberg, Germany Search for more papers by this author Isabelle Behm-Ansmant Isabelle Behm-Ansmant Max-Planck-Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author David Gatfield David Gatfield EMBL, Heidelberg, GermanyPresent address: University of Geneva, Quai Ernest-Ansermet, 1211-Geneva, Switzerland Search for more papers by this author Jan Rehwinkel Jan Rehwinkel EMBL, Heidelberg, Germany Search for more papers by this author Valérie Hilgers Valérie Hilgers EMBL, Heidelberg, Germany Search for more papers by this author Elisa Izaurralde Corresponding Author Elisa Izaurralde Max-Planck-Institute for Developmental Biology, Tübingen, Germany EMBL, Heidelberg, Germany Search for more papers by this author Author Information Isabelle Behm-Ansmant1, David Gatfield2, Jan Rehwinkel2, Valérie Hilgers2 and Elisa Izaurralde 1,2 1Max-Planck-Institute for Developmental Biology, Tübingen, Germany 2EMBL, Heidelberg, Germany *Corresponding author. Max-Planck-Institute for Developmental Biology, Spemannstrasse 35, Tübingen 72076, Germany. Tel.: +49 7071 601 1350; Fax: +49 7071 601 1353; E-mail: [email protected] The EMBO Journal (2007)26:1591-1601https://doi.org/10.1038/sj.emboj.7601588 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs with premature translation termination codons (PTCs). The mechanisms by which PTCs and natural stop codons are discriminated remain unclear. We show that the position of stops relative to the poly(A) tail (and thus of PABPC1) is a critical determinant for PTC definition in Drosophila melanogaster. Indeed, tethering of PABPC1 downstream of a PTC abolishes NMD. Conversely, natural stops trigger NMD when the length of the 3′ UTR is increased. However, many endogenous transcripts with exceptionally long 3′ UTRs escape NMD, suggesting that the increase in 3′ UTR length has co-evolved with the acquisition of features that suppress NMD. We provide evidence for the existence of 3′ UTRs conferring immunity to NMD. We also show that PABPC1 binding is sufficient for PTC recognition, regardless of cleavage or polyadenylation. The role of PABPC1 in NMD must go beyond that of providing positional information for PTC definition, because its depletion suppresses NMD under conditions in which translation efficiency is not affected. These findings reveal a conserved role for PABPC1 in mRNA surveillance. Introduction Nonsense-mediated mRNA decay (NMD) is a conserved mRNA quality control mechanism (surveillance) that ensures the fidelity of gene expression by detecting and degrading mRNAs containing premature translation termination codons (PTCs, nonsense codons). In this way, NMD safeguards cells from accumulating potentially deleterious truncated proteins (reviewed by Conti and Izaurralde 2005; Lejeune and Maquat, 2005; Amrani et al, 2006). The NMD pathway serves not only to degrade mRNAs containing PTCs (as a consequence of mutations or errors in transcription or mRNA processing), but also regulates the expression of about 3–10% of the transcriptome in Saccharomyces cerevisiae, Drosophila melanogaster and human cells. These natural NMD targets play a role in biological processes as diverse as transcription, cell proliferation, the cell cycle, telomere maintenance, cellular transport and organization, and metabolism (reviewed by Rehwinkel et al, 2006). NMD is triggered by premature translation termination, which leads to the assembly on the mRNA of a so-called surveillance complex. The surveillance complex comprises the conserved NMD effectors UPF1, UPF2 and UPF3, and couples the premature translation termination event to mRNA decay by interacting with both eukaryotic translation termination factors (i.e. eRF1 and eRF3) and with mRNA degradation enzymes (Conti and Izaurralde 2005; Lejeune and Maquat, 2005; Amrani et al, 2006). Stop codons are recognized as premature depending on their location relative to downstream sequence elements (DSEs) and associated proteins (Conti and Izaurralde 2005; Lejeune and Maquat, 2005; Amrani et al, 2006). In mammals, these downstream sequences are represented by exon–exon boundaries. Indeed, stop codons located at least 50–55 nt upstream of an exon–exon boundary are generally defined as premature, whereas most PTCs downstream of this point do not elicit decay (Nagy and Maquat, 1998). Exon–exon boundaries are marked by the exon junction complex (EJC), which is deposited during splicing 20–24 nt upstream of a splice junction (Le Hir et al, 2000). Current models for mammalian NMD postulate that UPF3 associates with the EJC within the nucleus and recruits UPF2 following the export of the mRNA to the cytoplasm. When translating ribosomes encounter a stop codon upstream of an EJC, the recruitment of UPF1 by translation release factors leads to an interaction with the UPF2 and UPF3 proteins bound to the downstream EJC, and thus to the assembly of the surveillance complex and to mRNA degradation (reviewed by Lejeune and Maquat, 2005). In S. cerevisiae and D. melanogaster, PTC recognition occurs independently of splicing, and different models have been proposed to explain what distinguishes premature from natural stops in these organisms. One model proposes that mRNAs harbor loosely defined DSEs with a function analogous to that of mammalian exon junctions (reviewed by Amrani et al, 2006). Alternative models suggest that a generic feature of the mRNA, such as the poly(A) tail, or a mark deposited during the cleavage and polyadenylation reaction could provide positional information to discriminate premature from natural stop codons (Hilleren and Parker, 1999; Muhlrad and Parker, 1999; Palaniswamy et al, 2006). Yet another model, the ‘faux 3′ UTR model’, proposes that premature translation termination is intrinsically aberrant because the stop codon is not in the appropriate context (Amrani et al, 2004, 2006). According to this model, natural 3′ UTRs are marked by a specific set of proteins that influence translation termination. Termination is efficient at natural stops because terminating ribosomes are able to interact with these 3′ UTR-bound proteins. In contrast, translation termination would be impaired or too slow at premature stops, because of the inability of the terminating ribosome to establish these interactions. In this case, the surveillance complex is assembled, leading to the rapid degradation of the mRNA (Amrani et al, 2004, 2006). In support of the ‘faux 3′ UTR model’, experiments in S. cerevisiae have shown that translation termination is aberrant at premature stop codons, and prematurely terminating ribosomes are not released efficiently (Amrani et al, 2004, 2006). This effect is abolished by flanking the nonsense codon with a normal 3′ UTR. Moreover, tethering the poly(A)-binding protein (Pab1p) downstream of a PTC, which is likely to mimic a normal 3′ UTR, leads to efficient translation termination and abolishes NMD (Amrani et al, 2004, 2006). This suggests that proximal Pab1p binding defines natural stops in S. cerevisiae. Consistently, most S. cerevisiae 3′ UTRs are homogeneous in length (ca. 100 nt; Graber et al, 1999), and aberrant transcripts with exceptionally long 3′ UTRs (due to errors in 3′-end processing) are regulated by NMD (Muhlrad and Parker, 1999). In multicellular organisms, 3′ UTR length distribution ranges from a few to several thousand nucleotides, raising the question of whether the faux 3′ UTR model could account for PTC recognition. We have investigated the mechanism of PTC recognition in D. melanogaster. We show that the cytoplasmic poly(A)-binding protein (PABPC1) provides positional information discriminating premature from natural stops in this organism. Consistently, natural stops can be made to trigger NMD by increasing the 3′ UTR length. However, a large proportion of naturally occurring transcripts with exceptionally long 3′ UTRs are not NMD substrates, suggesting that some of these 3′ UTRs may have evolved features to avoid NMD. Supporting this possibility, we show that 3′ UTRs specifying rapid decay also confer immunity to NMD. Finally, we demonstrate that depletion of PABPC1 inhibits NMD, revealing a new role for this protein in mRNA surveillance. Results Position effects of nonsense codons in D. melanogaster To gain further insights into the mechanism of PTC recognition in D. melanogaster, we introduced stop codons over the whole length of the D. melanogaster alcohol dehydrogenase (adh) open reading frame (ORF) C terminally fused to a V5 epitope. The adh-V5 ORF was cloned downstream of the actin 5C promoter and upstream of a polyadenylation site derived from SV40 (Figure 1A). The natural stop in this construct is located at codon 289. D. melanogaster Schneider cells (S2 cells) were transiently transfected with the reporters and a plasmid encoding a truncated version of adh (adhΔ), which served as a transfection control. In the experiments described below, the steady-state levels of the reporter mRNA were analyzed by Northern blot and normalized to those of the adhΔ mRNA. Figure 1.Boundary-dependent NMD in D. melanogaster. (A) Schematic representation of the adh reporters. Black boxes: exons; gray boxes: sequences derived from vector pAc5.1B; red box: V5 epitope; IN: intron. (B, C) S2 cells were transfected with vectors expressing adh-wt or the PTC reporters indicated above the lanes. A truncated version of adh (adhΔ) served as a transfection control. Transfected cells were then divided into two pools and treated with either GFP dsRNA (–) or UPF1 dsRNA (+). Total RNA samples were analyzed by Northern blot (B). The levels of the adh reporters were normalized to the levels of adhΔ mRNA. For each reporter, the normalized values obtained in UPF1-depleted cells were divided by those obtained in control cells (C). Mean values±s.d. of three independent experiments are shown. Kd: knockdown. Download figure Download PowerPoint Wild-type adh mRNA (adh-wt) was expressed at higher levels than adh mRNAs carrying PTCs at codon 64 or 83 (adh-64 or adh-83; Figure 1B, lane 1 versus lanes 3 and 5), as reported before (Gatfield et al, 2003). Similarly, the presence of PTCs at codons 113, 143, 173 and 203 led to a significant reduction of mRNA reporter levels, indicating that PTCs at codon 203 and upstream of it promote mRNA decay (Figure 1B, lanes 7, 9, 11 and 13). In contrast, mRNAs containing PTCs at codon 231 or 257 were expressed at levels comparable to those of the wild-type mRNA (Figure 1B, lanes 15 and 17), indicating that these PTCs are impaired in promoting mRNA degradation. To confirm that differences in reporter expression levels reflect genuine NMD, we analyzed whether these levels could be restored by depletion of the essential NMD effector UPF1. For the reporters carrying PTCs at codon 203 and upstream, depletion of UPF1 resulted in a three- to four-fold increase of mRNA levels (Figure 1B and C). The expression levels of adh-231 and adh-257 increased ca. 1.5-fold, whereas those of the wild-type mRNA did not change significantly (Figure 1B and C). These results indicate that the PTC-containing reporters are downregulated by NMD. They also show that there is a clear boundary effect rather than a polar effect of PTCs in this reporter, such that PTCs at codon 203 or upstream are efficient NMD triggers and those downstream cause modest NMD. In support of this, mRNAs with PTCs at codon 64 or 203 have identical half-lives (see Figure 2F). Thus, the extent of mRNA degradation depends on the position of the stop codon relative to downstream sequences. Figure 2.The ability of a PTC to trigger NMD depends on its distance from the 3′ UTR. (A) Schematic representation of the adh reporters. Symbols are as in Figure 1A. The distance of some PTCs to the polyadenylation site, as well as the positions of the insertion and deletion in adh-long and adh-short, respectively, are indicated. (B, C, E) S2 cells depleted of XRN1 were transfected with the reporters indicated above the lanes. RNA samples were analyzed by Northern blot (C, E) using a probe hybridizing to the 3′ UTR of the reporters (as shown in panel A). The levels of the 3′ decay intermediates were normalized to the levels of the full-length transcript (B). Mean values±s.d. of three independent experiments are shown. (D, F) S2 cells expressing the indicated adh reporters were treated with actinomycin D. Total RNA samples were collected at the indicated time points and analyzed by Northern blot. The levels of the adh reporters normalized to the levels of rp49 mRNA are plotted as a function of time. mRNA half-lives are indicated in brackets. Download figure Download PowerPoint The ability of a nonsense codon to trigger mRNA degradation is modulated by changing its distance to the 3′ UTR The positional effect of PTCs suggests that definition of a stop codon as premature depends on its position relative to either the 3′ UTR or to a DSE, which in our reporter should be between codons 203 and 231. In previous studies, we have shown that specific DSEs are unlikely to occur in D. melanogaster as PTC-containing mRNAs from bacterial origin are subject to NMD (Gatfield et al, 2003). If the distance to the 3′ UTR is the critical determinant for NMD, increasing the distance of PTC 231 or 257 to the 3′ UTR should increase the efficiency of these PTCs in eliciting mRNA decay. Conversely, decreasing the distance of PTCs 203 or 231 to the 3′ UTR should stabilize the transcript. To test this hypothesis, we inserted a 90 nt DNA fragment derived from the 3′ UTR of SV40 immediately downstream of PTC 257 and upstream of the natural stop. This DNA fragment has no stop codons, and in the wild-type context extends the adh ORF by 30 aa (Figure 2A, adh-long). In other words, relative to the poly(A) tail this insertion brings PTC 231 to the position occupied by PTC 203 in the normal context (Figure 2A). To measure NMD efficiency, we took advantage of the observation that NMD in D. melanogaster is initiated by endonucleolytic cleavage in the vicinity of the PTC. The resulting 3′ decay intermediate is digested exonucleolytically by the 5′ to 3′ exonuclease XRN1 (Gatfield and Izaurralde, 2004). Consequently, in cells depleted of XRN1, this fragment is stabilized and the extent of its accumulation relative to the full-length transcript is proportional to NMD efficiency. Using the relative accumulation of the 3′ decay intermediates in XRN1-depleted cells, we observed that increasing the distance of PTCs 231 or 257 to the 3′ UTR (adh-long reporter) triggered NMD more efficiently than in the normal context (Figure 2B and C, lanes 9 and 10 versus 4 and 5). These results were confirmed by measuring the half-lives of a transcript carrying a PTC at codon 231 in the normal or longer reporter (adh-231-normal or adh-231-long). The half-life of adh-231-normal mRNA was ca. 325 min (Figure 2D). When the distance of this PTC to the 3′ UTR was increased, its half-life was reduced to 66 min, comparable to that of a normal transcript with a PTC at codon 64 (Figure 2D). The insertion did not affect significantly the half-life of the adh wild-type reporter (Figure 2D). We next tested whether reducing the distance to the 3′ UTR could convert an efficient PTC into an inefficient trigger of NMD. To this end, we deleted a 78 nt DNA fragment between codons 231 and 257 (Figure 2A, adh-short). This deletion shifts PTC-203 relative to the 3′ end to the equivalent position of PTC-231 in the normal reporter (Figure 2A). Using the accumulation of the 3′ decay intermediates in XRN1-depleted cells, we observed that decreasing the distance of PTC 203 or PTC 231 to the 3′ UTR inhibited NMD (Figure 2B and E, lanes 7 and 8 versus 3 and 4, respectively), whereas the potential to elicit decay of the PTC 173 further upstream was unaltered (Figure 2B and E, lanes 5 and 9). These results were also confirmed by measuring mRNA half-lives. The half-life of adh-203 mRNA in the natural context was ca. 40 min. When the distance to the 3′ UTR was decreased, the half-life increased to more than 400 min (Figure 2F). The half-life of adh wild-type mRNA was not affected significantly by the deletion (data not shown). These results show that there is no DSE between codons 203 and 231, and clearly establish that it is the distance of the stop codon to the poly(A) tail (or other features of the 3′ UTR) that plays a critical role in NMD. Natural stops are redefined as premature when the length of the 3′ UTR is increased If the distance of the PTC to the poly(A) tail of the transcript plays a critical role in PTC recognition, one could also expect to convert a natural stop into a premature stop by increasing the length of the 3′ UTR. To test this possibility, we inserted a 198 nt cDNA fragment derived from the bacterial β-lactamase gene, upstream or downstream of the natural stop, thus doubling the length of the 3′ UTR (Figure 3A, ins. up or ins. down). Insertion of this fragment either upstream or downstream of the natural stop increased the efficiency of PTC-231 in triggering NMD (Figure 3B, C and F). This lends further support to the conclusion that specific DSEs within the ORF are unlikely to provide positional information for PTC-definition in D. melanogaster, as shown in Figure 2. Figure 3.Redefinition of a natural stop as premature by increasing the length of the 3′ UTR. (A) Schematic representation of the adh reporters having an insertion upstream (ins. up) or downstream (ins. down) of the natural stop (green boxes). Symbols are as in Figure 1A. (B–E) S2 cells were transfected with vectors expressing the indicated reporters. Plasmid adhΔ served as a transfection control. Transfected cells were treated with GFP (–) or UPF1 (+) dsRNAs. Panels B and D show representative Northern blots. In panels C and E, the levels of the adh mRNA reporters were normalized to the levels of adhΔ mRNA in three independent experiments. For each reporter, these ratios were set to 1 in control cells treated with GFP dsRNA (black bars). Mean values±s.d. are shown. (F) The levels of the 3′ decay intermediates accumulating in XRN1-depleted cells were normalized to the levels of the full-length transcript. Mean values±s.d. of three independent experiments are shown. Download figure Download PowerPoint Remarkably, out-of-frame insertion of the β-lactamase cDNA fragment downstream of the natural stop decreased the expression levels of the wild-type reporter in a UPF1-dependent manner (Figure 3D and E). That this reporter is indeed subject to NMD was confirmed by analyzing the accumulation of the corresponding 3′ decay intermediate in cells depleted of XRN1 (Figure 3F). Altogether, these results indicate that extending the 3′ UTR length does indeed result in the redefinition of the natural stop as premature. Binding of PABPC1 provides positional information for PTC-definition regardless of cleavage or polyadenylation The experiments described above provide strong evidence for a role of 3′ UTRs in PTC recognition: either the cleavage and polyadenylation reaction deposits a mark on the mRNA to provide positional information (see Palaniswamy et al, 2006) or PABPC1 itself plays a role in this process. To discriminate between these possibilities, we designed adh reporters in which the cleavage and polyadenylation signal is replaced either by the D. melanogaster histone H4 3′ stem–loop structure (Adamson and Price, 2003) or by a self-cleaving hammerhead ribozyme element (Dower et al, 2004; Figure 4A; adh-SL and adh-HhR reporters). Figure 4.Binding of PABPC1, regardless of cleavage and polyadenylation, is sufficient for PTC definition. (A) Schematic representation of the adh reporters in which the polyadenylation signal of SV40 is replaced by the histone H4 3′ stem–loop (SL) or a self-cleavable hammerhead ribozyme (HhR). Symbols are as in Figure 1A. The insertion of a poly(A) stretch is indicated by a green box. (B–E) S2 cells transfected with the reporters indicated above the panels were treated with GFP (–) or UPF1 (+) dsRNAs. Panels B and E show representative Northern blots. In panels C and D, the levels of the adh reporters were normalized to the levels of adhΔ mRNA in three independent experiments and analyzed as described in Figures 3C and E. (F, G) S2 cells were transfected with the indicated adh reporters. Total protein and RNA samples were analyzed by Western and Northern blots, respectively, and quantitated in three independent experiments. Download figure Download PowerPoint When the 3′ end of the transcript was generated by the histone 3′-end-processing machinery, a reporter bearing a PTC at codon 64 was no longer regulated by NMD, as judged by the observation that the steady-state levels of the reporter did not change upon UPF1 depletion (64-SL reporter, Figure 4B, lanes 7 and 8; Figure 4C). Similar results were obtained for the reporter containing the hammerhead ribozyme (adh-Hhr reporter). Indeed, the expression levels of adh-64-HhR mRNA (carrying a PTC at codon 64) were unaffected by UPF1 depletion (Figure 4D and E, lanes 3 and 4). Importantly, wild-type mRNAs whose 3′ ends are generated by ribozyme cleavage or by the histone 3′-end-processing machinery are exported to the cytoplasm and translated, as judged by the detection of the ADH-V5 fusion protein by Western blot (Figure 4F and G; Duvel et al, 2002; Dower et al, 2004). Furthermore, when we compared the expression levels of the ADH-V5 protein with the corresponding mRNA levels, we saw that the translation efficiency of these non-polyadenylated mRNAs was comparable to that of the mRNAs having a poly(A) tail (Figure 4G). This indicates that the absence of NMD for PTC-containing, unadenylated mRNAs cannot be attributed to reduced translation efficiency. To determine whether susceptibility to NMD could be restored by binding of PABPC1, we followed two approaches. First, we inserted a stretch of DNA-encoded adenosine residues immediately upstream of the histone stem–loop structure or of the ribozyme element (Figure 4A; adh-(A)45-SL and adh-(A)75-Hhr). It has previously been shown that a stretch of at least 45 adenosines is sufficient for PABPC1 binding in vivo (Dower et al, 2004). The artificial poly(A) tails rescued the ability of PTC-64 to promote NMD in both cases (Figures 4B–E). The insertion of the poly(A) stretch increased steady-state mRNA levels (Figure 4B and E), but had no effect on the translation efficiency of the corresponding wild-type mRNAs (Figure 4F and G). This observation supports the conclusion that the failure of PTC-64 to trigger mRNA decay in the reporters lacking a stretch of adenosine residues is not caused by inefficient translation. In the second approach, we used a tethering assay based on the high-affinity interaction between the RNA-binding peptide derived from the bacteriophage-λN protein (λN-peptide) and an RNA hairpin known as BoxB element (Gehring et al, 2003). A chimeric protein consisting of the λN-peptide fused to D. melanogaster PABPC1 was coexpressed with adh reporter mRNAs in which five BoxB elements were inserted immediately upstream of the hammerhead ribozyme. Tethering of λN-PABPC1 increased mRNA levels and partially rescued the ability of PTC 64 to promote NMD (Supplementary Figure 1) as with the insertion of an artificial poly(A) tail. In summary, these results indicate that PABPC1 is sufficient to provide positional information for PTC recognition, regardless of 3′-end cleavage and polyadenylation. PABPC1 tethered downstream of a PTC abolishes NMD We next tested whether tethering PABPC1 downstream of a PTC could convert it into an NMD-insensitive stop. To this end, we coexpressed λN-PABPC1 with adh reporter mRNAs in which four BoxB elements were inserted at codon 117 (Figure 5A, 4BoxB). To control for nonspecific effects that may arise by overexpressing λN-PABPC1, we coexpressed untagged PABPC1 with the reporters. Figure 5.Tethering of PABPC1 suppresses NMD in a polar manner. (A) Schematic representation of the adh-4BoxB reporters. Symbols are as in Figure 1A. White box: 4BoxB tethering sites. (B, C) S2 cells were transfected with the adh-4BoxB reporters, adhΔ and plasmids encoding the λN-peptide, λN-PABPC1 or untagged PABPC1 as indicated. Total RNA samples were analyzed by Northern blot (B). The levels of the adh reporters were normalized to the levels of adhΔ mRNA. For each reporter, these levels were set to 1 in cells expressing the λN-peptide alone (C, white bars). Mean values±s.d. of three independent experiments are shown. (D) S2 cells expressing Adh-113-4BoxB in the presence of the λN-peptide or λN-PABPC1 were treated with actinomycin D. Samples were collected at the indicated time points and analyzed by Northern blot. The levels of the adh mRNA normalized to the levels of rp49 mRNA are plotted as a function of time. The half-lives of the adh mRNA are indicated. (E–G) S2 cells were transfected with adh-wt-4BoxB or adh-113-4BoxB and plasmids encoding the proteins indicated above the lanes. Samples were analyzed as described in panel C. In panel E, the expression levels of PABPC1 and PABPN1 were analyzed by Western blot. ADH-V5 served as a transfection control. Download figure Download PowerPoint Tethering of PABPC1 downstream of PTCs 64, 83 and 113 suppressed NMD in a polar manner: the closer the PTC to the tethering site, the better the suppression by PABPC1 (Figure 5B and C). Consistently, the half-life of adh-113 mRNA increased when PABPC1 was tethered immediately downstream of this PTC (Figure 5D). In contrast, PABPC1 did not suppress NMD for PTCs located downstream of the tethering site (PTCs 143 and 173; Figure 5B and C). To examine whether the effects of PABPC1 are specific, we also tested the effect of tethering the nuclear poly(A)-binding protein 1 (PABPN1) downstream of adh-113. In contrast to PABPC1, which suppresses NMD, PABPN1 has no effect, despite both proteins being expressed at comparable levels (Figure 5E–G). The results obtained for the adh reporter were validated with a second reporter based on the bacterial chloramphenicol acetyl transferase (CAT) gene (Figure 6). Indeed, tethering of PABPC1 downstream of codon 129 suppresses NMD triggered by PTCs upstream, but not downstream, of the tethering site (Figure 6A–C). In agreement with these observations, the half-life of CAT-126 increased when PABPC1 was tethered immediately downstream (Figure 6D and E). We conclude that, as in S. cerevisiae (Amrani et al, 2004, 2006), tethering of PABPC1 downstream of a PTC in D. melanogaster abolishes NMD in a polar manner. Figure 6.Tethered PABPC1 suppresses NMD. (A) Schematic representation of the CAT-4BoxB reporters. Symbols are as in Figure 5A. (B, C) S2 cells were transfected with vectors expressing CAT-4BoxB reporters and plasmids encoding the proteins indicated above the lanes. Total RNA samples were analyzed by Northern blot (B). In panel C, the levels of the CAT reporters were analyzed as described in Figure 5C. (D, E) S2 cells expressing CAT-126-4BoxB in the presence of the λN-peptide or λN-PABPC1 were treated with actinomycin D. Samples were analyzed as described in Figure 5D. Download figure Download PowerPoint The polar effect of tethered PABPC1 is in contrast with the boundary effect observed when the PTC is moved in the context of a natural poly(A) tail (Figure 1). This suggests that tethering of PABPC1 may not fully reproduce its normal function when it binds directly to poly(A) tail of the mRNA. Indeed, only four PABPC1 molecules can be tethered to the reporter; they do not contact the RNA directly, and may not interact with each other as they normally would (Bernstein and Ross, 1989). Most endogenous transcripts with long 3′ UTRs are not regulated by NMD The results described above indicate that PABPC1 plays a critical role in NMD but do not determine completely whether it is the distance to PABPC1 alone that distinguishes a premature from a normal stop or whether sequence context also plays a role. If the distance to the PABPC1-binding site were alone sufficient to discriminate normal versus premature stops, transcripts with 3′ UTRs longer than average would be expected to be regulated by NMD. In a previous study in which we identified endogenous NMD targets by expression profiling of cells depleted of NMD factors, we reported that NMD targets are not enriched significantly with transcripts having long 3′ UTRs (Rehwinkel et al, 2005). Indeed, detectable transcripts in S2 cells have an average 3′ UTR length of 450 nt, whereas the average 3′ UTR length of NMD targets is 520 nt. When we analyzed the length distribution of 3′ UTRs in windows of 500 nt, we saw that NMD targets are slightly enri
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