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• W2102284002 abstract "The selective degradation of mRNAs by the nonsense-mediated decay pathway is a quality control process with important consequences for human disease. From initial studies using RNA hairpin-tagged mRNAs for purification of messenger ribonucleoproteins assembled on transcripts with HIV-1 3′ untranslated region (3′UTR) sequences, we uncover a two-step mechanism for Upf1-dependent degradation of mRNAs with long 3′UTRs. We demonstrate that Upf1 associates with mRNAs in a 3′UTR length-dependent manner and is highly enriched on transcripts containing 3′UTRs known to elicit NMD. Surprisingly, Upf1 recruitment and subsequent RNA decay can be antagonized by retroviral RNA elements that promote translational readthrough. By modulating the efficiency of translation termination, recognition of long 3′UTRs by Upf1 is uncoupled from the initiation of decay. We propose a model for 3′UTR length surveillance in which equilibrium binding of Upf1 to mRNAs precedes a kinetically distinct commitment to RNA decay. The selective degradation of mRNAs by the nonsense-mediated decay pathway is a quality control process with important consequences for human disease. From initial studies using RNA hairpin-tagged mRNAs for purification of messenger ribonucleoproteins assembled on transcripts with HIV-1 3′ untranslated region (3′UTR) sequences, we uncover a two-step mechanism for Upf1-dependent degradation of mRNAs with long 3′UTRs. We demonstrate that Upf1 associates with mRNAs in a 3′UTR length-dependent manner and is highly enriched on transcripts containing 3′UTRs known to elicit NMD. Surprisingly, Upf1 recruitment and subsequent RNA decay can be antagonized by retroviral RNA elements that promote translational readthrough. By modulating the efficiency of translation termination, recognition of long 3′UTRs by Upf1 is uncoupled from the initiation of decay. We propose a model for 3′UTR length surveillance in which equilibrium binding of Upf1 to mRNAs precedes a kinetically distinct commitment to RNA decay. Upf1 assembles into mRNPs in a 3′UTR length-dependent manner Retroviral readthrough-promoting elements inhibit Upf1 recruitment and mRNA decay Rare readthrough events allow Upf1 accumulation in mRNPs but antagonize mRNA decay Upf1 3′UTR length sensing is coupled to a kinetically distinct commitment to decay The nonsense-mediated decay (NMD) machinery executes important regulatory and quality control functions by targeting specific classes of messenger RNAs (mRNAs) for degradation (Chang et al., 2007Chang Y.-F. Imam J.S. Wilkinson M.F. The nonsense-mediated decay RNA surveillance pathway.Annu. Rev. Biochem. 2007; 76: 51-74Crossref PubMed Scopus (882) Google Scholar). In addition to degrading transcripts containing premature termination codons (PTCs) resulting from mutation or rearrangement of genomic DNA or defects in mRNA biogenesis, the pathway is also responsible for regulating between 1% and 10% of all genes in diverse eukaryotes (He et al., 2003He F. Li X. Spatrick P. Casillo R. Dong S. Jacobson A. Genome-wide analysis of mRNAs regulated by the nonsense-mediated and 5′ to 3′ mRNA decay pathways in yeast.Mol. Cell. 2003; 12: 1439-1452Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, Mendell et al., 2004Mendell J.T. Sharifi N.A. Meyers J.L. Martinez-Murillo F. Dietz H.C. Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise.Nat. Genet. 2004; 36: 1073-1078Crossref PubMed Scopus (603) Google Scholar, Rehwinkel et al., 2005Rehwinkel J. Letunic I. Raes J. Bork P. Izaurralde E. Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets.RNA. 2005; 11: 1530-1544Crossref PubMed Scopus (201) Google Scholar, Wittmann et al., 2006Wittmann J. Hol E.M. Jäck H.-M. hUPF2 silencing identifies physiologic substrates of mammalian nonsense-mediated mRNA decay.Mol. Cell. Biol. 2006; 26: 1272-1287Crossref PubMed Scopus (176) Google Scholar, Weischenfeldt et al., 2008Weischenfeldt J. Damgaard I. Bryder D. Theilgaard-Mönch K. Thoren L.A. Nielsen F.C. Jacobsen S.E.W. Nerlov C. Porse B.T. NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements.Genes Dev. 2008; 22: 1381-1396Crossref PubMed Scopus (181) Google Scholar). Transcripts preferentially targeted by NMD include those with PTCs encoded by alternatively spliced exons, introns downstream of the termination codon (TC), long 3′ untranslated regions (3′UTRs), or upstream open reading frames (uORFs; reviewed in Nicholson et al., 2010Nicholson P. Yepiskoposyan H. Metze S. Zamudio Orozco R. Kleinschmidt N. Mühlemann O. Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors.Cell. Mol. Life Sci. 2010; 67: 677-700Crossref PubMed Scopus (240) Google Scholar, Rebbapragada and Lykke-Andersen, 2009Rebbapragada I. Lykke-Andersen J. Execution of nonsense-mediated mRNA decay: what defines a substrate?.Curr. Opin. Cell Biol. 2009; 21: 394-402Crossref PubMed Scopus (210) Google Scholar). A characteristic that is common to many NMD decay substrates is an extended distance from the terminating ribosome to the mRNA 3′ end (i.e., 3′UTR length). Degradation of aberrant mRNAs by NMD can affect the progression of many human genetic disorders, an estimated one-third of which derive from PTCs (Kuzmiak and Maquat, 2006Kuzmiak H.A. Maquat L.E. Applying nonsense-mediated mRNA decay research to the clinic: progress and challenges.Trends Mol. Med. 2006; 12: 306-316Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). In addition, shortening of 3′UTRs has been proposed to relax regulation of mRNA stability and translation, promoting cellular transformation (Sandberg et al., 2008Sandberg R. Neilson J.R. Sarma A. Sharp P.A. Burge C.B. Proliferating cells express mRNAs with shortened 3′ untranslated regions and fewer microRNA target sites.Science. 2008; 320: 1643-1647Crossref PubMed Scopus (912) Google Scholar, Wang et al., 2008Wang E.T. Sandberg R. Luo S. Khrebtukova I. Zhang L. Mayr C. Kingsmore S.F. Schroth G.P. Burge C.B. Alternative isoform regulation in human tissue transcriptomes.Nature. 2008; 456: 470-476Crossref PubMed Scopus (3200) Google Scholar, Mayr and Bartel, 2009Mayr C. Bartel D.P. Widespread shortening of 3'UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells.Cell. 2009; 138: 673-684Abstract Full Text Full Text PDF PubMed Scopus (1021) Google Scholar). These findings underscore the importance of understanding the mechanisms by which 3′UTR length is sensed in the process of mRNA quality control. The well-conserved superfamily I RNA helicase Upf1 is a crucial component of the core NMD machinery. Like other RNA helicases, Upf1 exhibits nonspecific but robust RNA binding activity modulated by ATP binding and hydrolysis (Weng et al., 1998Weng Y. Czaplinski K. Peltz S.W. ATP is a cofactor of the Upf1 protein that modulates its translation termination and RNA binding activities.RNA. 1998; 4: 205-214PubMed Google Scholar, Bhattacharya et al., 2000Bhattacharya A. Czaplinski K. Trifillis P. He F. Jacobson A. Peltz S.W. Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay.RNA. 2000; 6: 1226-1235Crossref PubMed Scopus (137) Google Scholar). Though the functional roles of Upf1's ATPase and helicase activities are unclear, mutations that abolish its ATPase activity prevent NMD (Weng et al., 1996aWeng Y. Czaplinski K. Peltz S.W. Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein.Mol. Cell. Biol. 1996; 16: 5477-5490Crossref PubMed Scopus (174) Google Scholar, Weng et al., 1996bWeng Y. Czaplinski K. Peltz S.W. Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover.Mol. Cell. Biol. 1996; 16: 5491-5506Crossref PubMed Scopus (131) Google Scholar, Sun et al., 1998Sun X. Perlick H.A. Dietz H.C. Maquat L.E. A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsense-containing mRNAs in mammalian cells.Proc. Natl. Acad. Sci. USA. 1998; 95: 10009-10014Crossref PubMed Scopus (157) Google Scholar). In addition, Upf1 participates in a network of interactions with additional factors proposed to mediate its association with mRNA targets and regulate a cycle of Upf1 phosphorylation and dephosphorylation required for establishment of translational repression and recruitment of RNA decay enzymes (reviewed in Nicholson et al., 2010Nicholson P. Yepiskoposyan H. Metze S. Zamudio Orozco R. Kleinschmidt N. Mühlemann O. Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors.Cell. Mol. Life Sci. 2010; 67: 677-700Crossref PubMed Scopus (240) Google Scholar; see below). Within the context of a long 3′UTR, additional mRNA features and protein components of mRNPs can promote or inhibit decay. For example, the exon-junction complex (EJC), a multiprotein assembly deposited at exon-exon junctions in the process of splicing, acts through Upf1 to strongly activate decay (Le Hir et al., 2000Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions.EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (677) Google Scholar, Le Hir et al., 2001Le Hir H. Gatfield D. Izaurralde E. Moore M.J. The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay.EMBO J. 2001; 20: 4987-4997Crossref PubMed Scopus (575) Google Scholar, Kim et al., 2001Kim V.N. Kataoka N. Dreyfuss G. Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex.Science. 2001; 293: 1832-1836Crossref PubMed Scopus (240) Google Scholar, Lykke-Andersen et al., 2001Lykke-Andersen J. Shu M.D. Steitz J.A. Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1.Science. 2001; 293: 1836-1839Crossref PubMed Scopus (294) Google Scholar). The competition between Upf1 and cytoplasmic poly(A)-binding protein 1 (PABPC1) for binding to the translation release factors eRF1 and eRF3 has been proposed to be a crucial factor in the decision to decay diverse transcripts (Ivanov et al., 2008Ivanov P.V. Gehring N.H. Kunz J.B. Hentze M.W. Kulozik A.E. Interactions between UPF1, eRFs, PABP and the exon junction complex suggest an integrated model for mammalian NMD pathways.EMBO J. 2008; 27: 736-747Crossref PubMed Scopus (226) Google Scholar, Singh et al., 2008Singh G. Rebbapragada I. Lykke-Andersen J. A competition between stimulators and antagonists of Upf complex recruitment governs human nonsense-mediated mRNA decay.PLoS Biol. 2008; 6: e111Crossref PubMed Scopus (225) Google Scholar). Upf1 binding to release factors at the terminating ribosome stimulates phosphorylation of Upf1 by the SMG-1 kinase, translational repression, and recruitment of decay factors (Kashima et al., 2006Kashima I. Yamashita A. Izumi N. Kataoka N. Morishita R. Hoshino S. Ohno M. Dreyfuss G. Ohno S. Binding of a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay.Genes Dev. 2006; 20: 355-367Crossref PubMed Scopus (398) Google Scholar, Isken et al., 2008Isken O. Kim Y.K. Hosoda N. Mayeur G.L. Hershey J.W.B. Maquat L.E. Upf1 phosphorylation triggers translational repression during nonsense-mediated mRNA decay.Cell. 2008; 133: 314-327Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, Cho et al., 2009Cho H. Kim K.M. Kim Y.K. Human proline-rich nuclear receptor coregulatory protein 2 mediates an interaction between mRNA surveillance machinery and decapping complex.Mol. Cell. 2009; 33: 75-86Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Conversely, binding of PABPC1 to release factors is proposed to preserve transcript stability and translational competence. In support of this model, artificial tethering approaches and alterations in 3′UTR structure designed to mimic 3′UTR shortening by bringing PABPC1 in proximity to the termination codon can suppress Upf1-dependent decay (Amrani et al., 2004Amrani N. Ganesan R. Kervestin S. Mangus D.A. Ghosh S. Jacobson A. A faux 3′-UTR promotes aberrant termination and triggers nonsense-mediated mRNA decay.Nature. 2004; 432: 112-118Crossref PubMed Scopus (350) Google Scholar, Behm-Ansmant et al., 2007Behm-Ansmant I. Gatfield D. Rehwinkel J. Hilgers V. Izaurralde E. A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay.EMBO J. 2007; 26: 1591-1601Crossref PubMed Scopus (150) Google Scholar, Eberle et al., 2008Eberle A.B. Stalder L. Mathys H. Orozco R.Z. Mühlemann O. Posttranscriptional gene regulation by spatial rearrangement of the 3′ untranslated region.PLoS Biol. 2008; 6: e92Crossref PubMed Scopus (206) Google Scholar, Ivanov et al., 2008Ivanov P.V. Gehring N.H. Kunz J.B. Hentze M.W. Kulozik A.E. Interactions between UPF1, eRFs, PABP and the exon junction complex suggest an integrated model for mammalian NMD pathways.EMBO J. 2008; 27: 736-747Crossref PubMed Scopus (226) Google Scholar, Silva et al., 2008Silva A.L. Ribeiro P. Inácio A. Liebhaber S.A. Romão L. Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay.RNA. 2008; 14: 563-576Crossref PubMed Scopus (109) Google Scholar). Here, we use affinity purification of hairpin-tagged mRNAs to isolate and characterize endogenously assembled mRNP complexes. With this approach, we show that Upf1 assembles into mRNPs in a 3′UTR length-dependent manner. Upf1 copurifies to some extent with all transcripts tested but is highly enriched on mRNAs containing 3′UTRs derived from known NMD targets. The preferential association of Upf1 with mRNAs containing NMD-sensitive 3′UTRs is not affected by inhibition of translation and NMD. Together with our finding that the efficiency of Upf1 coimmunoprecipitation with 3′UTR-derived RNase H cleavage products correlates with fragment length, these observations suggest a direct role for Upf1 in 3′UTR length sensing. To further investigate the in vivo dynamics of 3′UTR length surveillance and decay, we use retroviral elements to induce translational readthrough of NMD-triggering termination codons. Surprisingly, periodic readthrough events can reduce steady-state Upf1 association with transcripts containing long 3′UTRs and robustly inhibit NMD. Moreover, we show that rare readthrough events permit steady-state Upf1 accumulation in mRNPs but prevent initiation of mRNA decay. Our data inform a model in which equilibrium binding of Upf1 senses 3′UTR length and establishes an RNP state primed for decay. The identification of potential decay targets by Upf1 is coupled to a subsequent commitment to decay, the rate of which is dependent on other aspects of mRNP structure and composition. Furthermore, our data indicate that the decision to decay takes place over a kinetic interval corresponding to many translation termination events. This separation between 3′UTR length sensing and initiation of decay provides a mechanism to prevent aberrant degradation of normal RNAs and presents an opportunity for transcripts to evade cellular mRNA surveillance. Retroviruses may exploit this opportunity by inducing translational readthrough or frameshifting to periodically disrupt the recognition of viral mRNAs as potential decay substrates. To better understand cellular mRNA biogenesis and decay, we have developed a generalizable technique for purification and characterization of endogenously assembled mRNP complexes. In this approach, we singly tag mRNAs with the naturally occurring Pseudomonas phage 7 coat protein (PP7CP) binding site, a 25 nucleotide (nt) stably folding hairpin (Figure 1A ; Lim and Peabody, 2002Lim F. Peabody D.S. RNA recognition site of PP7 coat protein.Nucleic Acids Res. 2002; 30: 4138-4144Crossref PubMed Scopus (61) Google Scholar). Tagged RNAs are transiently or stably expressed in appropriate mammalian cell lines, allowing progression through endogenous RNA processing pathways. RNPs assembled on the tagged RNAs are then purified from extracts using a version of the PP7CP tagged with tandem Staphylococcus aureus protein A domains. Previously, a similar method was used to isolate complexes associated with several noncoding RNAs (Hogg and Collins, 2007aHogg J.R. Collins K. Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control.Genes Dev. 2007; 21: 3067-3072Crossref PubMed Scopus (52) Google Scholar, Hogg and Collins, 2007bHogg J.R. Collins K. RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation.RNA. 2007; 13: 868-880Crossref PubMed Scopus (110) Google Scholar). In the process of adapting this methodology to the purification of mRNPs, we found that the use of traditional agarose-based resins afforded inefficient purification of tagged mRNP complexes. In contrast, nonporous magnetic resins allowed purification of tagged mRNAs to near homogeneity following a single step of purification (Figures 1A and 1C; additional data not shown). Recent work in our laboratory has shown that HIV-1 3′LTR sequences play a crucial role in the regulation of viral mRNA biogenesis (Valente and Goff, 2006Valente S.T. Goff S.P. Inhibition of HIV-1 gene expression by a fragment of hnRNP U.Mol. Cell. 2006; 23: 597-605Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, Valente et al., 2009Valente S.T. Gilmartin G.M. Mott C. Falkard B. Goff S.P. Inhibition of HIV-1 replication by eIF3f.Proc. Natl. Acad. Sci. USA. 2009; 106: 4071-4078Crossref PubMed Scopus (30) Google Scholar). To identify proteins specifically associated with HIV 3′LTR sequences, we constructed a series of PP7-tagged RNAs containing the GFP open reading frame and alternative 3′UTRs (Figure 1B; see below). In our initial experiments, we used a version of the HIV 3′LTR containing a deletion in the U3 region (ΔU3 LTR). The bovine growth hormone polyadenylation (bGH pA) element of the pcDNA3.1 vector was used as a control, aiding discrimination of proteins specifically bound to HIV 3′LTR sequence-containing RNPs. Silver staining of complexes purified from whole-cell extracts of transiently transfected 293T cells revealed that, as expected, each tagged RNA associates with a large number of proteins (Figure 1C and data not shown). Mock purifications from extracts lacking tagged RNAs exhibited very few contaminating proteins, indicating that the vast majority of the proteins visible by silver staining were isolated via their association with tagged mRNPs. Many components of the purified mRNPs are found in all complexes purified, including general translation factors, ribosomes, hnRNP proteins, and other proteins that associate with common mRNA features (Figure 1C and data not shown). Tandem mass spectrometry of gel slices excised from HIV ΔU3 LTR and bGH copurifying material identified four peptides derived from the Upf1 protein in the ΔU3 LTR sample but none in the bGH control sample (Table S1 available online). Immunoblotting of PP7-purified RNPs confirmed that Upf1 was enriched on transcripts containing HIV 3′LTR sequences, using immunoblotting for PABPC1 as a control for RNP recovery (Figure 2A ). We detected Upf1 in association with RNAs containing the bGH pA element, but at much lower levels than those copurifying with RNAs containing the ΔU3 LTR sequence. Still higher levels of Upf1 were isolated using a full-length LTR (FLTR) comprising intact U3, R, and U5 LTR segments from the pNL4.3 reference HIV genome (Figure 1B and Figure 2A). In agreement with observations of human Upf1 cosedimentation with bulk polysomes and coimmunoprecipitation with diverse mRNAs (Pal et al., 2001Pal M. Ishigaki Y. Nagy E. Maquat L.E. Evidence that phosphorylation of human Upfl protein varies with intracellular location and is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway.RNA. 2001; 7: 5-15Crossref PubMed Scopus (117) Google Scholar, Hosoda et al., 2005Hosoda N. Kim Y.K. Lejeune F. Maquat L.E. CBP80 promotes interaction of Upf1 with Upf2 during nonsense-mediated mRNA decay in mammalian cells.Nat. Struct. Mol. Biol. 2005; 12: 893-901Crossref PubMed Scopus (108) Google Scholar), we find that Upf1 associates to some degree with all RNAs tested (Figure 2A, Figure 3A and data not shown). Importantly, we additionally observe substantial transcript-specific enrichment of Upf1 in mRNPs (see below).Figure 3Upf1 Preferentially Associates with Transcripts Containing 3′UTRs Known to Trigger NMDShow full caption(A) PP7-tagged GFP mRNAs containing the indicated 3′UTRs were transiently expressed in 293T cells and subjected to affinity purification. Proteins present in whole-cell extracts and purified RNPs were detected by immunoblotting with antibodies against endogenous Upf1, PABPC1, SMG-1, and Upf2. (Bottom) RNA was isolated from small fractions of extracts and purified material and analyzed by northern blotting. See also Figures S1A and S1B.(B) Upf1 recruitment is insensitive to cycloheximide treatment. 293T cells transiently transfected with PP7-tagged GFP mRNAs containing the TRAM1 or SMG5 3′UTRs were treated (+) or not treated (−) with cycloheximide for 4 hr prior to cell harvest and throughout extract preparation and affinity purification. Immunoblotting and northern blotting were performed as in (A). Inhibition of NMD by cycloheximide and persistence of Upf1 recruitment under conditions of translation inhibition by puromycin and 5′-proximal hairpins are illustrated in Figures S1C–S1F.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) PP7-tagged GFP mRNAs containing the indicated 3′UTRs were transiently expressed in 293T cells and subjected to affinity purification. Proteins present in whole-cell extracts and purified RNPs were detected by immunoblotting with antibodies against endogenous Upf1, PABPC1, SMG-1, and Upf2. (Bottom) RNA was isolated from small fractions of extracts and purified material and analyzed by northern blotting. See also Figures S1A and S1B. (B) Upf1 recruitment is insensitive to cycloheximide treatment. 293T cells transiently transfected with PP7-tagged GFP mRNAs containing the TRAM1 or SMG5 3′UTRs were treated (+) or not treated (−) with cycloheximide for 4 hr prior to cell harvest and throughout extract preparation and affinity purification. Immunoblotting and northern blotting were performed as in (A). Inhibition of NMD by cycloheximide and persistence of Upf1 recruitment under conditions of translation inhibition by puromycin and 5′-proximal hairpins are illustrated in Figures S1C–S1F. To address the specificity of Upf1 association with RNPs containing HIV 3′LTR sequences, we first created tagged RNA constructs in which the ΔU3 or full-length LTR elements were cloned in the antisense orientation, with the bGH pA element provided downstream to ensure proper 3′ end maturation. The requirement for an additional 3′ end-processing element caused the antisense 3′UTRs to be ∼200 nt longer than their sense equivalents (Figure 2B, see northern blot). As above, we observed increasing Upf1 copurification with the bGH, ΔU3, and FLTR RNAs, respectively (Figure 2B). Surprisingly, the levels of Upf1 associated with the antisense LTR-containing RNAs were slightly higher than with the corresponding sense 3′UTRs. Thus, the observed recruitment of Upf1 to LTR-containing RNAs was not dependent on primary sequence or structural features. Instead, our data suggested that Upf1 accumulation in mRNPs might be dictated by 3′UTR length. Current models suggest that 3′UTR length is a crucial determinant of NMD susceptibility (Mühlemann, 2008Mühlemann O. Recognition of nonsense mRNA: towards a unified model.Biochem. Soc. Trans. 2008; 36: 497-501Crossref PubMed Scopus (39) Google Scholar, Rebbapragada and Lykke-Andersen, 2009Rebbapragada I. Lykke-Andersen J. Execution of nonsense-mediated mRNA decay: what defines a substrate?.Curr. Opin. Cell Biol. 2009; 21: 394-402Crossref PubMed Scopus (210) Google Scholar), but the mechanism by which 3′UTR length is sensed remains unclear. To test the hypothesis that Upf1 associates with transcripts in a 3′UTR length-dependent manner, we generated a series of 5′ PP7-tagged RNAs consisting of the GFP ORF fused to the HIV FLTR, such that the fragment of the HIV nef ORF contained in the LTR was in frame with the GFP ORF (Figure 2C). This series of constructs contains single termination codons at ∼100 nt intervals, starting with the original GFP termination codon and ending with the nef termination codon ∼400 nt downstream. In this way, we varied 3′UTR length by making only one (ablation of the GFP TC) or two (ablation of the GFP TC combined with introduction of a new in-frame TC) point mutations to the RNA primary sequence. Using these constructs, we found that Upf1 copurification with tagged mRNAs increased with 3′UTR length (Figure 2C). The relationship between Upf1 copurification and 3′UTR length was strikingly linear, consistent with sequence-nonspecific recognition of long 3′UTRs by Upf1 (Figure 2D). Our observations suggested that Upf1 might accomplish 3′UTR length sensing by associating with 3′UTRs. To better understand the basis for 3′UTR length-dependent accumulation of Upf1 in mRNPs, we used RNase H and a series of oligonucleotides directed against HIV 3′LTR sequence to site-specifically cleave 5′-tagged GFP-FLTR mRNAs at sites ∼7, ∼211, and ∼305 nucleotides downstream of the GFP TC. Following RNase H digestion, we immunoprecipitated endogenous Upf1 and assayed mRNA recovery by northern blotting using a probe against HIV 3′LTR sequence. The RNase H cleavage conditions were designed to leave a substantial fraction of the mRNAs intact, allowing the use of full-length mRNAs as recovery controls. FLTR-containing mRNAs were recovered with an antibody against Upf1, but not nonspecific control goat IgG (Figure 2E and Figure S1A ). Consistent with our observations above, the efficiency of 3′UTR fragment coimmunoprecipitation increased with RNA length (Figures 2E and 2F). These data suggest that Upf1 association along the length of 3′UTRs accounts for the observed 3′UTR length-dependent accumulation in mRNPs. Our observation that Upf1 association correlates with 3′UTR length mirrors prior findings that 3′UTR extension causes progressive transcript destabilization in mammalian cells (Bühler et al., 2006Bühler M. Steiner S. Mohn F. Paillusson A. Mühlemann O. EJC-independent degradation of nonsense immunoglobulin-μ mRNA depends on 3′ UTR length.Nat. Struct. Mol. Biol. 2006; 13: 462-464Crossref PubMed Scopus (173) Google Scholar, Eberle et al., 2008Eberle A.B. Stalder L. Mathys H. Orozco R.Z. Mühlemann O. Posttranscriptional gene regulation by spatial rearrangement of the 3′ untranslated region.PLoS Biol. 2008; 6: e92Crossref PubMed Scopus (206) Google Scholar, Singh et al., 2008Singh G. Rebbapragada I. Lykke-Andersen J. A competition between stimulators and antagonists of Upf complex recruitment governs human nonsense-mediated mRNA decay.PLoS Biol. 2008; 6: e111Crossref PubMed Scopus (225) Google Scholar). To assess the functional significance of the enrichment of Upf1 on specific mRNAs, we used a series of long 3′UTRs shown by Singh and colleagues (2008) to either promote or evade decay. As representative NMD-insensitive long 3′UTRs, we used the human CRIPT1 (1515 nt) and TRAM1 (1494 nt) 3′UTRs. To model targets of 3′UTR length-dependent NMD, we used the human SMG5 3′UTR (1342 nt) and an artificial 3′UTR comprising a portion of the GAPDH ORF and the GAPDH 3′UTR (GAP; 846 nt). As above, we transiently transfected 293T cells with tagged RNA constructs containing model 3′UTRs and isolated mRNPs from whole-cell extracts with PP7CP. Immunoblotting of purified RNPs revealed that Upf1 association strongly correlated with NMD sensitivity (Figure 3A). Very low levels of Upf1 copurified with transcripts containing the NMD-insensitive CRIPT1 and TRAM1 3′UTRs. In contrast, transcripts containing the intronless GAP and SMG5 3′UTRs copurified high levels of Upf1, with the SMG5 3′UTR-containing mRNAs showing the greatest Upf1 recruitment. Likewise, antibodies against Upf1 coimmunoprecipitated mRNAs containing the GAP and SMG5 3′UTRs at higher efficiencies than mRNAs containing the CRIPT and TRAM 3′UTRs (Figure S1A). We did not observe the NMD factors SMG-1 or Upf2 in PP7-purified mRNPs, despite robust detection of the proteins in whole-cell extracts used for purification (Figure 3A). In similar experiments, comparable levels of Upf1 copurified with mRNAs containing the intronless GAP 3′UTR and a version of the GAP 3′UTR containing the adenovirus major-late intron (GAP AdML; Figure S1B). This observation suggests that 3′UTR length is a more significant determinant of Upf1 association than the presence of a spliced intron downstream of the TC. Together, these findings indicate that the extent of Upf1 association with a transcript is diagnostic of its NMD susceptibility, consistent with previous experiments in yeast, C. elegans, and human cells (Johansson et al., 2007Johansson M.J.O. He F. Spatrick P. Li C. Jacobson A. Association of yeast Upf1p with direct substrates of the NMD pathway.Proc. Natl. Acad. Sci. USA. 2007; 104: 20872-20877Crossref PubMed Scopus (68) Google Scholar" @default.
• W2102284002 created "2016-06-24" @default.
• W2102284002 creator A5007813632 @default.
• W2102284002 creator A5031225867 @default.
• W2102284002 date "2010-10-01" @default.
• W2102284002 modified "2023-10-12" @default.
• W2102284002 title "Upf1 Senses 3′UTR Length to Potentiate mRNA Decay" @default.
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