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The RNA-binding protein PTBP1 promotes ATPase-dependent dissociation of the RNA helicase UPF1 to protect transcripts from nonsense-mediated mRNA decay

2020; Elsevier BV; Volume: 295; Issue: 33 Linguagem: Inglês

10.1074/jbc.ra120.013824

ISSN

1083-351X

Autores

Sarah E. Fritz, Soumya Ranganathan, Clara D. Wang, J. Robert Hogg,

Tópico(s)

RNA modifications and cancer

Resumo

The sequence-specific RNA-binding proteins PTBP1 (polypyrimidine tract–binding protein 1) and HNRNP L (heterogeneous nuclear ribonucleoprotein L) protect mRNAs from nonsense-mediated decay (NMD) by preventing the UPF1 RNA helicase from associating with potential decay targets. Here, by analyzing in vitro helicase activity, dissociation of UPF1 from purified mRNPs, and transcriptome-wide UPF1 RNA binding, we present the mechanistic basis for inhibition of NMD by PTBP1. Unlike mechanisms of RNA stabilization that depend on direct competition for binding sites among protective RNA-binding proteins and decay factors, PTBP1 promotes displacement of UPF1 already bound to potential substrates. Our results show that PTBP1 directly exploits the tendency of UPF1 to release RNA upon ATP binding and hydrolysis. We further find that UPF1 sensitivity to PTBP1 is coordinated by a regulatory loop in domain 1B of UPF1. We propose that the UPF1 regulatory loop and protective proteins control kinetic proofreading of potential NMD substrates, presenting a new model for RNA helicase regulation and target selection in the NMD pathway. The sequence-specific RNA-binding proteins PTBP1 (polypyrimidine tract–binding protein 1) and HNRNP L (heterogeneous nuclear ribonucleoprotein L) protect mRNAs from nonsense-mediated decay (NMD) by preventing the UPF1 RNA helicase from associating with potential decay targets. Here, by analyzing in vitro helicase activity, dissociation of UPF1 from purified mRNPs, and transcriptome-wide UPF1 RNA binding, we present the mechanistic basis for inhibition of NMD by PTBP1. Unlike mechanisms of RNA stabilization that depend on direct competition for binding sites among protective RNA-binding proteins and decay factors, PTBP1 promotes displacement of UPF1 already bound to potential substrates. Our results show that PTBP1 directly exploits the tendency of UPF1 to release RNA upon ATP binding and hydrolysis. We further find that UPF1 sensitivity to PTBP1 is coordinated by a regulatory loop in domain 1B of UPF1. We propose that the UPF1 regulatory loop and protective proteins control kinetic proofreading of potential NMD substrates, presenting a new model for RNA helicase regulation and target selection in the NMD pathway. Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved pathway that fulfills quality control and regulatory functions by targeting specific mRNAs for degradation. As a quality control mechanism, NMD eliminates aberrant transcripts undergoing premature translation termination events caused by genetic lesions or errors in mRNA processing or translation (1Kishor A. Fritz S.E. Hogg J.R. Nonsense-mediated mRNA decay: the challenge of telling right from wrong in a complex transcriptome.Wiley Interdisciplinary Reviews: RNA. 2019; 10 (31131562): e154810.1002/wrna.1548Crossref PubMed Scopus (54) Google Scholar). In addition, 5–10% of apparently normal mRNAs are subject to NMD, a broad target specificity that cells have harnessed to control gene expression programs in development, stress responses, immunity, and other important contexts (2Goetz A.E. Wilkinson M. Stress and the nonsense-mediated RNA decay pathway.Cell. Mol. Life Sci. 2017; 74 (28503708): 3509-353110.1007/s00018-017-2537-6Crossref PubMed Scopus (48) Google Scholar, 3Kurosaki T. Popp M.W. Maquat L.E. Quality and quantity control of gene expression by nonsense-mediated mRNA decay.Nat. Rev. Mol. Cell Biol. 2019; 20 (30992545): 406-42010.1038/s41580-019-0126-2Crossref PubMed Scopus (287) Google Scholar). The central coordinator of the NMD pathway is the RNA helicase UPF1 (4Kim Y.K. Maquat L.E. UPFront and center in RNA decay: UPF1 in nonsense-mediated mRNA decay and beyond.RNA. 2019; 25 (30655309): 407-42210.1261/rna.070136.118Crossref PubMed Scopus (97) Google Scholar), a superfamily 1 RNA helicase with the capacity to translocate 5′ to 3′ on single-stranded nucleic acids, unwind duplexes, and disrupt RNA–protein interactions (5Czaplinski K. Weng Y. Hagan K.W. Peltz S.W. Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation.RNA. 1995; 1 (7489520): 610-623PubMed Google Scholar, 6Bhattacharya 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 (10999600): 1226-123510.1017/s1355838200000546Crossref PubMed Scopus (143) Google Scholar, 7Fiorini F. Bagchi D. Le Hir H. Croquette V. Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities.Nat. Commun. 2015; 6 (26138914): 758110.1038/ncomms8581Crossref PubMed Scopus (83) Google Scholar). UPF1 has been described to engage in direct interactions with translation termination factors, ribosomes, mRNAs, nucleases, and other NMD pathway components to promote decay (1Kishor A. Fritz S.E. Hogg J.R. Nonsense-mediated mRNA decay: the challenge of telling right from wrong in a complex transcriptome.Wiley Interdisciplinary Reviews: RNA. 2019; 10 (31131562): e154810.1002/wrna.1548Crossref PubMed Scopus (54) Google Scholar). Through these interactions, UPF1 can integrate multiple streams of information about the translation and composition of mRNPs to determine which mRNAs should be degraded. Despite the elucidation of a dense network of protein–protein interactions involved in decay, the process by which NMD targets are selected remains to be fully understood. In addition to serving as a hub for assembly of decay enzymes and other NMD factors, UPF1 participates in target discrimination by directly binding to mRNAs (8Hurt J.A. Robertson A.D. Burge C.B. Global analyses of UPF1 binding and function reveal expanded scope of nonsense-mediated mRNA decay.Genome Res. 2013; 23 (23766421): 1636-165010.1101/gr.157354.113Crossref PubMed Scopus (162) Google Scholar, 9Zünd D. Gruber A.R. Zavolan M. Mühlemann O. Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3′ UTRs.Nat. Struct. Mol. Biol. 2013; 20 (23832275): 936-94310.1038/nsmb.2635Crossref PubMed Scopus (112) Google Scholar). UPF1 binds RNA with high affinity but low sequence specificity, allowing it to comprehensively surveil the transcriptome (8Hurt J.A. Robertson A.D. Burge C.B. Global analyses of UPF1 binding and function reveal expanded scope of nonsense-mediated mRNA decay.Genome Res. 2013; 23 (23766421): 1636-165010.1101/gr.157354.113Crossref PubMed Scopus (162) Google Scholar, 9Zünd D. Gruber A.R. Zavolan M. Mühlemann O. Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3′ UTRs.Nat. Struct. Mol. Biol. 2013; 20 (23832275): 936-94310.1038/nsmb.2635Crossref PubMed Scopus (112) Google Scholar, 10Chakrabarti S. Jayachandran U. Bonneau F. Fiorini F. Basquin C. Domcke S. Le Hir H. Conti E. Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2.Mol. Cell. 2011; 41 (21419344): 693-70310.1016/j.molcel.2011.02.010Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). UPF1 preferentially accumulates on 3′-UTRs because of a combination of nonspecific RNA binding and active displacement from coding sequences during translation (9Zünd D. Gruber A.R. Zavolan M. Mühlemann O. Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3′ UTRs.Nat. Struct. Mol. Biol. 2013; 20 (23832275): 936-94310.1038/nsmb.2635Crossref PubMed Scopus (112) Google Scholar, 11Hogg J.R. Goff S.P. Upf1 senses 3′ UTR length to potentiate mRNA decay.Cell. 2010; 143 (21029861): 379-38910.1016/j.cell.2010.10.005Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). In experiments using reporter mRNAs and transcriptome-wide studies, UPF1 binding correlates with 3′-UTR length, providing a mechanism by which long 3′-UTRs can be recognized by the NMD pathway (8Hurt J.A. Robertson A.D. Burge C.B. Global analyses of UPF1 binding and function reveal expanded scope of nonsense-mediated mRNA decay.Genome Res. 2013; 23 (23766421): 1636-165010.1101/gr.157354.113Crossref PubMed Scopus (162) Google Scholar, 11Hogg J.R. Goff S.P. Upf1 senses 3′ UTR length to potentiate mRNA decay.Cell. 2010; 143 (21029861): 379-38910.1016/j.cell.2010.10.005Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 12Baker S.L. Hogg J.R. A system for coordinated analysis of translational readthrough and nonsense-mediated mRNA decay.PLoS One. 2017; 12 (28323884): e017398010.1371/journal.pone.0173980Crossref PubMed Scopus (21) Google Scholar, 13Kurosaki T. Li W. Hoque M. Popp M.W.-L. Ermolenko D.N. Tian B. Maquat L.E. A post-translational regulatory switch on UPF1 controls targeted mRNA degradation.Genes Dev. 2014; 28 (25184677): 1900-191610.1101/gad.245506.114Crossref PubMed Scopus (107) Google Scholar). Although the degree of UPF1 binding to an mRNA is correlated with decay susceptibility, UPF1 associates with many mRNAs that are not substrates for NMD (8Hurt J.A. Robertson A.D. Burge C.B. Global analyses of UPF1 binding and function reveal expanded scope of nonsense-mediated mRNA decay.Genome Res. 2013; 23 (23766421): 1636-165010.1101/gr.157354.113Crossref PubMed Scopus (162) Google Scholar, 9Zünd D. Gruber A.R. Zavolan M. Mühlemann O. Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3′ UTRs.Nat. Struct. Mol. Biol. 2013; 20 (23832275): 936-94310.1038/nsmb.2635Crossref PubMed Scopus (112) Google Scholar, 11Hogg J.R. Goff S.P. Upf1 senses 3′ UTR length to potentiate mRNA decay.Cell. 2010; 143 (21029861): 379-38910.1016/j.cell.2010.10.005Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 13Kurosaki T. Li W. Hoque M. Popp M.W.-L. Ermolenko D.N. Tian B. Maquat L.E. A post-translational regulatory switch on UPF1 controls targeted mRNA degradation.Genes Dev. 2014; 28 (25184677): 1900-191610.1101/gad.245506.114Crossref PubMed Scopus (107) Google Scholar). These observations support a two-step model in which the length-dependent accumulation of UPF1 on 3′-UTRs increases the probability of decay but does not represent a commitment step. Instead, at least one kinetically distinct step must be fulfilled for degradation to occur. Thus, an important point of control in NMD target selection may be regulation of UPF1 residence time on mRNAs (11Hogg J.R. Goff S.P. Upf1 senses 3′ UTR length to potentiate mRNA decay.Cell. 2010; 143 (21029861): 379-38910.1016/j.cell.2010.10.005Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 14Lee S.R. Pratt G.A. Martinez F.J. Yeo G.W. Lykke-Andersen J. Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity.Mol. Cell. 2015; 59 (26253027): 413-42510.1016/j.molcel.2015.06.036Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 15Durand S. Franks T.M. Lykke-Andersen J. Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay.Nat. Commun. 2016; 7 (27511142): 1243410.1038/ncomms12434Crossref PubMed Scopus (44) Google Scholar). UPF1 persistence on potential NMD substrates can be modulated by ATP binding and hydrolysis (6Bhattacharya 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 (10999600): 1226-123510.1017/s1355838200000546Crossref PubMed Scopus (143) Google Scholar, 10Chakrabarti S. Jayachandran U. Bonneau F. Fiorini F. Basquin C. Domcke S. Le Hir H. Conti E. Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2.Mol. Cell. 2011; 41 (21419344): 693-70310.1016/j.molcel.2011.02.010Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 13Kurosaki T. Li W. Hoque M. Popp M.W.-L. Ermolenko D.N. Tian B. Maquat L.E. A post-translational regulatory switch on UPF1 controls targeted mRNA degradation.Genes Dev. 2014; 28 (25184677): 1900-191610.1101/gad.245506.114Crossref PubMed Scopus (107) Google Scholar, 14Lee S.R. Pratt G.A. Martinez F.J. Yeo G.W. Lykke-Andersen J. Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity.Mol. Cell. 2015; 59 (26253027): 413-42510.1016/j.molcel.2015.06.036Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Weng 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 (8816461): 5477-549010.1128/mcb.16.10.5477Crossref PubMed Scopus (180) Google Scholar, 17Weng 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 (9570320): 205-214PubMed Google Scholar, 18Cheng Z. Muhlrad D. Lim M.K. Parker R. Song H. Structural and functional insights into the human Upf1 helicase core.EMBO J. 2007; 26 (17159905): 253-26410.1038/sj.emboj.7601464Crossref PubMed Scopus (114) Google Scholar, 19Gowravaram M. Bonneau F. Kanaan J. Maciej V.D. Fiorini F. Raj S. Croquette V. Le Hir H. Chakrabarti S. A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner.Nucleic Acids Res. 2018; 46 (29378013): 2648-265910.1093/nar/gky040Crossref PubMed Scopus (17) Google Scholar, 20Serdar L.D. Whiteside D.L. Baker K.E. ATP hydrolysis by UPF1 is required for efficient translation termination at premature stop codons.Nat. Commun. 2016; 7 (28008922): 1402110.1038/ncomms14021Crossref PubMed Scopus (31) Google Scholar). The helicase core of UPF1 consists of two tandem RecA-like domains that function to bind RNA and hydrolyze ATP (10Chakrabarti S. Jayachandran U. Bonneau F. Fiorini F. Basquin C. Domcke S. Le Hir H. Conti E. Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2.Mol. Cell. 2011; 41 (21419344): 693-70310.1016/j.molcel.2011.02.010Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 18Cheng Z. Muhlrad D. Lim M.K. Parker R. Song H. Structural and functional insights into the human Upf1 helicase core.EMBO J. 2007; 26 (17159905): 253-26410.1038/sj.emboj.7601464Crossref PubMed Scopus (114) Google Scholar, 19Gowravaram M. Bonneau F. Kanaan J. Maciej V.D. Fiorini F. Raj S. Croquette V. Le Hir H. Chakrabarti S. A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner.Nucleic Acids Res. 2018; 46 (29378013): 2648-265910.1093/nar/gky040Crossref PubMed Scopus (17) Google Scholar). In addition, UPF1 harbors two extension domains (domains 1B and 1C) that protrude from RecA1. An 11-amino acid regulatory loop in domain 1B specifically reduces the affinity of UPF1 for RNA in the presence of ATP (10Chakrabarti S. Jayachandran U. Bonneau F. Fiorini F. Basquin C. Domcke S. Le Hir H. Conti E. Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2.Mol. Cell. 2011; 41 (21419344): 693-70310.1016/j.molcel.2011.02.010Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 18Cheng Z. Muhlrad D. Lim M.K. Parker R. Song H. Structural and functional insights into the human Upf1 helicase core.EMBO J. 2007; 26 (17159905): 253-26410.1038/sj.emboj.7601464Crossref PubMed Scopus (114) Google Scholar, 19Gowravaram M. Bonneau F. Kanaan J. Maciej V.D. Fiorini F. Raj S. Croquette V. Le Hir H. Chakrabarti S. A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner.Nucleic Acids Res. 2018; 46 (29378013): 2648-265910.1093/nar/gky040Crossref PubMed Scopus (17) Google Scholar). Mechanistically, this regulatory loop protrudes into the RNA-binding channel in the ATP-bound state, weakening the affinity of UPF1 for RNA in the presence of ATP (5Czaplinski K. Weng Y. Hagan K.W. Peltz S.W. Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation.RNA. 1995; 1 (7489520): 610-623PubMed Google Scholar, 6Bhattacharya 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 (10999600): 1226-123510.1017/s1355838200000546Crossref PubMed Scopus (143) Google Scholar, 10Chakrabarti S. Jayachandran U. Bonneau F. Fiorini F. Basquin C. Domcke S. Le Hir H. Conti E. Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2.Mol. Cell. 2011; 41 (21419344): 693-70310.1016/j.molcel.2011.02.010Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 13Kurosaki T. Li W. Hoque M. Popp M.W.-L. Ermolenko D.N. Tian B. Maquat L.E. A post-translational regulatory switch on UPF1 controls targeted mRNA degradation.Genes Dev. 2014; 28 (25184677): 1900-191610.1101/gad.245506.114Crossref PubMed Scopus (107) Google Scholar, 16Weng 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 (8816461): 5477-549010.1128/mcb.16.10.5477Crossref PubMed Scopus (180) Google Scholar, 17Weng 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 (9570320): 205-214PubMed Google Scholar, 18Cheng Z. Muhlrad D. Lim M.K. Parker R. Song H. Structural and functional insights into the human Upf1 helicase core.EMBO J. 2007; 26 (17159905): 253-26410.1038/sj.emboj.7601464Crossref PubMed Scopus (114) Google Scholar, 19Gowravaram M. Bonneau F. Kanaan J. Maciej V.D. Fiorini F. Raj S. Croquette V. Le Hir H. Chakrabarti S. A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner.Nucleic Acids Res. 2018; 46 (29378013): 2648-265910.1093/nar/gky040Crossref PubMed Scopus (17) Google Scholar). We have recently reported that cells prevent UPF1 recognition of many long 3′-UTRs by recruiting specific RNA-binding proteins to the vicinity of termination codons. Two protective proteins, PTBP1 (polypyrimidine tract-binding protein 1) and hnRNP L (heterogeneous nuclear ribonucleoprotein L) reshape NMD target specificity, together shielding hundreds to thousands of human 3′-UTRs from NMD (21Ge Z. Quek B.L. Beemon K.L. Hogg J.R. Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway.Elife. 2016; 5 (26744779): e1115510.7554/eLife.11155Crossref PubMed Google Scholar, 22Kishor A. Ge Z. Hogg J.R. hnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma.EMBO J. 2019; 38 (30530525): e9912810.15252/embj.201899128Crossref PubMed Scopus (35) Google Scholar). PTBP1 and hnRNP L prefer CU- and CA-rich sequences, respectively, but share an evolutionary origin and protein architecture, suggesting that they may inhibit UPF1 association through a common mechanism (23Oberstrass F.C. Auweter S.D. Erat M. Hargous Y. Henning A. Wenter P. Reymond L. Amir-Ahmady B. Pitsch S. Black D.L. Allain F.H.-T. Structure of PTB bound to RNA: specific binding and implications for splicing regulation.Science. 2005; 309 (16179478): 2054-205710.1126/science.1114066Crossref PubMed Scopus (331) Google Scholar, 24Blatter M. Dunin-Horkawicz S. Grishina I. Maris C. Thore S. Maier T. Bindereif A. Bujnicki J.M. Allain F.H.-T. The signature of the five-stranded vRRM fold defined by functional, structural and computational analysis of the hnRNP L protein.J. Mol. Biol. 2015; 427 (26051023): 3001-302210.1016/j.jmb.2015.05.020Crossref PubMed Scopus (21) Google Scholar, 25Hui J. Stangl K. Lane W.S. Bindereif A. HnRNP L stimulates splicing of the eNOS gene by binding to variable-length CA repeats.Nat. Struct. Biol. 2003; 10 (12447348): 33-3710.1038/nsb875Crossref PubMed Scopus (131) Google Scholar). Experiments using reporter mRNAs as well as genome-wide studies support a role for the protective proteins in preventing steady-state accumulation of UPF1 in mRNPs. However, the nonspecific nature of UPF1 association with 3′-UTRs raises the question of how sequence-specific RNA-binding proteins PTBP1 and hnRNP L are able to prevent UPF1 association with potential target 3′-UTRs. Here we present the molecular mechanism that explains how binding of PTBP1 to specific 3′-UTR sites can antagonize nonspecific UPF1 binding. Rather than a direct competition for binding sites, PTBP1 stimulates the dissociation of UPF1 from potential target mRNAs. Inhibition of UPF1 by PTBP1 is governed by the regulatory loop in UPF1 domain 1B that reduces UPF1 affinity for RNA in the presence of ATP (18Cheng Z. Muhlrad D. Lim M.K. Parker R. Song H. Structural and functional insights into the human Upf1 helicase core.EMBO J. 2007; 26 (17159905): 253-26410.1038/sj.emboj.7601464Crossref PubMed Scopus (114) Google Scholar, 19Gowravaram M. Bonneau F. Kanaan J. Maciej V.D. Fiorini F. Raj S. Croquette V. Le Hir H. Chakrabarti S. A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner.Nucleic Acids Res. 2018; 46 (29378013): 2648-265910.1093/nar/gky040Crossref PubMed Scopus (17) Google Scholar). We propose that UPF1 selects potential decay targets through a kinetic proofreading mechanism regulated by the domain 1B regulatory loop and mRNP components, presenting a new model for RNA helicase regulation and target selection by the NMD pathway. To define the mechanism by which PTBP1 and hnRNP L prevent UPF1 association with potential NMD targets, we developed a fluorescence-based assay of UPF1 translocation. A target RNA substrate was designed with a 5′ 60-nt CA-rich RNA segment predicted to allow efficient UPF1 but not PTBP1 binding, a 30-nt CU-rich sequence previously shown to be bound by PTBP1 with ∼50 nm affinity, and an 18-nt 3′ oligohybridization region (Fig. 1A) (26Amir-Ahmady B. Boutz P.L. Markovtsov V. Phillips M.L. Black D.L. Exon repression by polypyrimidine tract binding protein.RNA. 2005; 11 (15840818): 699-71610.1261/rna.2250405Crossref PubMed Scopus (90) Google Scholar). Annealing the in vitro transcribed RNA to a complementary oligonucleotide labeled at the 5′ end with a fluorophore created a labeled, duplexed substrate (Fig. 1A). Unwinding reactions were performed in the presence of an excess of trap-strand oligonucleotide labeled at the 3′ end with a Black Hole quencher (BHQ). Upon addition of ATP, UPF1 activity resulted in a decrease in fluorescence caused by displacement of the labeled oligonucleotide and subsequent quenching by the trap strand (Fig. 1A). For our experiments, we analyzed the activity of a UPF1 protein containing the helicase core (amino acids 295–914) but lacking the autoinhibitory CH domain (UPF1ΔCH) (Fig. S1A) (10Chakrabarti S. Jayachandran U. Bonneau F. Fiorini F. Basquin C. Domcke S. Le Hir H. Conti E. Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2.Mol. Cell. 2011; 41 (21419344): 693-70310.1016/j.molcel.2011.02.010Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 27Fiorini F. Bonneau F. Le Hir H. Biochemical characterization of the RNA helicase UPF1 involved in nonsense-mediated mRNA decay.Methods Enzymol. 2012; 511 (22713324): 255-27410.1016/B978-0-12-396546-2.00012-7Crossref PubMed Scopus (15) Google Scholar). Highly purified UPF1ΔCH (Fig. S1B) exhibited strong unwinding activity, displacing the duplexed oligonucleotide in an ATP-dependent manner (Fig. 1B). In contrast, preincubation of PTBP1 with the RNA impaired the ability of UPF1ΔCH to unwind the 3′ duplex in a dose-dependent manner (Fig. 1B). We observed similar behavior using a second RNA substrate with a 20-nt single-stranded region upstream of the PTBP1-binding site (Fig. S1C), confirming that PTBP1-mediated inhibition was not specific to a particular substrate. UPF1ΔCH exerts sufficient force to displace streptavidin from a 3′ biotinylated RNA substrate (7Fiorini F. Bagchi D. Le Hir H. Croquette V. Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities.Nat. Commun. 2015; 6 (26138914): 758110.1038/ncomms8581Crossref PubMed Scopus (83) Google Scholar), leading us to hypothesize a specific effect of PTBP1 on UPF1 helicase activity rather than a mechanical barrier to translocation. In addition to verifying that the UPF1ΔCH protein used here for unwinding assays could displace streptavidin from biotin (Fig. S1D), we tested whether the Pseudomonas phage 7 coat protein (PP7cp), which has nanomolar affinity for its cognate RNA-binding site (28Lim F. Downey T.P. Peabody D.S. Translational repression and specific RNA binding by the coat protein of the Pseudomonas phage PP7.J. Biol. Chem. 2001; 276 (11306589): 22507-2251310.1074/jbc.M102411200Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), was able to antagonize UPF1 translocation. Despite its tight association with its RNA-binding site (Fig. S1E), PP7cp failed to disrupt UPF1 unwinding (Fig. 1C and Fig. S1F). Based on previous characterization of UPF1 and PTBP1 RNA-binding properties (10Chakrabarti S. Jayachandran U. Bonneau F. Fiorini F. Basquin C. Domcke S. Le Hir H. Conti E. Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2.Mol. Cell. 2011; 41 (21419344): 693-70310.1016/j.molcel.2011.02.010Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 26Amir-Ahmady B. Boutz P.L. Markovtsov V. Phillips M.L. Black D.L. Exon repression by polypyrimidine tract binding protein.RNA. 2005; 11 (15840818): 699-71610.1261/rna.2250405Crossref PubMed Scopus (90) Google Scholar), the ∼90 nt of single-stranded RNA provided upstream of the duplex region of the helicase assay substrates should provide ample room for concurrent binding of UPF1 and PTBP1. However, it remained possible that the inhibition of UPF1 activity by PTBP1 was due to either prevention of initial binding or disruption of UPF1 translocation. To begin to elucidate the underlying mechanism, we next asked whether PTBP1-mediated impairment of UPF1ΔCH unwinding activity depended on the order in which the two proteins were added to the reaction. Consistent with a mechanism in which PTBP1 inhibits UPF1 translocation rather than initial binding, PTBP1 added simultaneously with or following prebinding of UPF1ΔCH to the assay substrate inhibited unwinding to an extent indistinguishable from that achieved by prebound PTBP1 (Fig. 1D). The experiments described thus far were performed under multiple turnover conditions, meaning that UPF1 has the ability to cycle between bound and unbound states on substrate RNAs. To further address the possibility that the observed inhibition was due to reduced rates of rebinding of UPF1, we conducted unwinding assays under single-turnover conditions in which an excess of trap ssDNA was added to capture UPF1 after its dissociation from the helicase assay substrate. As observed under multiple-turnover conditions, PTBP1 impaired the ability of UPF1ΔCH to unwind the 3′ duplex under single turnover conditions (Fig. 1E). Taken together, these results support a model in which PTBP1 inhibits UPF1 5′ to 3′ translocation, in a manner independent of its ability to prevent initial UPF1 binding to RNA. The observations that UPF1 displaces streptavidin from 3′ biotinylated substrates and is insensitive to the high-affinity PP7cp-hairpin interaction suggested that a mechanism in which PTBP1 serves only as a mechanical roadblock was unlikely to explain our findings. Therefore, we next sought to further test the possibility that PTBP1 binding to the target RNA inhibits UPF1 5′ to 3′ translocation by serving as a physical barrier to translocation. To this end, we used a DNA substrate containing a pyrimidine-rich sequence at the 5′ end. Previous in vitro studies have found that PTBP1 retains a preference for pyrimidines in ssDNA (29Brunel F. Zakin M.M. Buc H. Buckle M. The polypyrimidine tract binding (PTB) protein interacts with single-stranded DNA in a sequence-specific manner.Nucleic Acids Res. 1996; 24 (8649976): 1608-161510.1093/nar/24.9.1608Crossref PubMed Scopus (27) Google Scholar). PTBP1 bound in this position would allow UPF1 binding to the remaining 40 nt of ssDNA upstream of the substrate duplex region (Fig. 2A). Despite the lack of PTBP1-binding sites between the duplex and the single-stranded UPF1-binding region, PTBP1 exerted strong, dosage-dependent inhibition of UPF1ΔCH unwinding on this substrate (Fig. 2A). This result provides further evidence that PTBP1 inhibits UPF1 activity without preventing substrate binding or providing a mere physical roadblock to translocation. To further test the requirement for high-affinity PTBP1 binding to the nucleic acid substrate for UPF1 inhibition, we next used a target DNA that harbored a 70-nt 5′ UPF1-binding region devoid of polypyrimidine sequences and an 18-nt 3′ oligo hybridization region. Binding assays using this DNA substrate confirmed that UPF1ΔCH but not PTBP1 detectably associated with the DNA substrate (Fig. S2). Despite lacking stable binding to the DNA substrate, PTBP1 impaired the ability of UPF1ΔCH to unwind the target DNA (Fig. 2B), albeit at a higher concentration than observed with the RNA substrate (Fig. 1A) or the DNA substrate with a 5′ pyrimidine-rich sequence (Fig. 2A). Together, these data suggest that the biochemical mechanism of UPF1 inhibition by PTBP1 does not strictly require PTBP1 to engage the UPF1-bound nucleic acid with high affinity or in a position that impairs the path of UPF1 translocation. However, consistent with previous findings that enrichment of PTBP1 near NMD-sensitive stop codons is required for biologically meaningful protection (21Ge Z. Quek B.L. Beemon K.L. Hogg J.R. Polypyrimidine tract binding protein 1 protects mR

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