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• W2076580369 abstract "Polypyrimidine tract binding (PTB) protein is a regulator of alternative pre-mRNA splicing, and also stimulates the initiation of translation dependent on many viral internal ribosome entry segments/sites (IRESs). It has four RNA-binding domains (RBDs), but although the contacts with many IRESs have been mapped, the orientation of binding (i.e., which RBD binds to which site in the IRES) is unknown. To answer this question, 16 derivatives of PTB1, each with a single cysteine flanking the RNA-binding surface in an RBD, were constructed and used in directed hydroxyl radical probing with the encephalomyocarditis virus IRES. The results, together with mass spectrometry data on the stoichiometry of PTB binding to different IRES derivatives, show that the minimal IRES binds a single PTB in a unique orientation, with RBD1 and RBD2 binding near the 3′ end, and RBD3 contacting the 5′ end, thereby constraining and stabilizing the three-dimensional structural fold of the IRES. Polypyrimidine tract binding (PTB) protein is a regulator of alternative pre-mRNA splicing, and also stimulates the initiation of translation dependent on many viral internal ribosome entry segments/sites (IRESs). It has four RNA-binding domains (RBDs), but although the contacts with many IRESs have been mapped, the orientation of binding (i.e., which RBD binds to which site in the IRES) is unknown. To answer this question, 16 derivatives of PTB1, each with a single cysteine flanking the RNA-binding surface in an RBD, were constructed and used in directed hydroxyl radical probing with the encephalomyocarditis virus IRES. The results, together with mass spectrometry data on the stoichiometry of PTB binding to different IRES derivatives, show that the minimal IRES binds a single PTB in a unique orientation, with RBD1 and RBD2 binding near the 3′ end, and RBD3 contacting the 5′ end, thereby constraining and stabilizing the three-dimensional structural fold of the IRES. Soon after its discovery as a nuclear protein with high affinity for intronic pyrimidine-rich tracts in pre-mRNA, polypyrimidine tract binding (PTB) protein, also known as hnRNPI, was recognized to be an important regulator of alternative splicing (reviewed in Valcarcel and Gebauer, 1997Valcarcel J. Gebauer F. Post-transcriptional regulation: the dawn of PTB.Curr. Biol. 1997; 7: R705-R708Abstract Full Text Full Text PDF PubMed Google Scholar, Sawicka et al., 2008Sawicka K. Bushell M. Spriggs K.A. Willis A.E. Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein.Biochem. Soc. Trans. 2008; 36: 641-647Crossref PubMed Scopus (210) Google Scholar). It was noted that although hnRNPI is predominantly located in the nucleus, there is considerably more in the cytoplasm than is typical of hnRNPs, implying that it is probably a shuttling protein (Ghetti et al., 1992Ghetti A. Pinolroma S. Michael W.M. Morandi C. Dreyfuss G. hnRNP-I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs.Nucleic Acids Res. 1992; 20: 3671-3678Crossref PubMed Scopus (263) Google Scholar). Indeed, an influence of PTB on several different cytoplasmic events has been reported, for example the localization of certain mRNAs and the stability of others (reviewed in Sawicka et al., 2008Sawicka K. Bushell M. Spriggs K.A. Willis A.E. Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein.Biochem. Soc. Trans. 2008; 36: 641-647Crossref PubMed Scopus (210) Google Scholar). However, the best known cytoplasmic function is the stimulation of translation initiation dependent on picornaviral internal ribosome entry sites/segments (IRESs), which was discovered very soon after PTB was first characterized (reviewed in Jackson and Kaminski, 1995Jackson R.J. Kaminski A. Internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond.RNA. 1995; 1: 985-1000PubMed Google Scholar). Subsequently, PTB has been reported to stimulate the activity of many cellular mRNA IRESs, and it has even been suggested that PTB may be a general trans-acting factor for IRES-dependent initiation (Sawicka et al., 2008Sawicka K. Bushell M. Spriggs K.A. Willis A.E. Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein.Biochem. Soc. Trans. 2008; 36: 641-647Crossref PubMed Scopus (210) Google Scholar). The PTB requirement for picornavirus IRESs varies quite widely according to the species. It is absolutely required for all of the Type I (entero- and rhinovirus) IRESs that have been tested (Hunt and Jackson, 1999Hunt S.L. Jackson R.J. Polypyrimidine-tract binding protein (PTB) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 RNA.RNA. 1999; 5: 344-359Crossref PubMed Scopus (125) Google Scholar), but Type II picornavirus IRESs show more variability: the foot-and-mouth disease virus (FMDV) IRES shows strong dependency on PTB (Niepmann, 1996Niepmann M. Porcine polypyrimidine tract-binding protein stimulates translation initiation at the internal ribosome entry site of foot-and-mouth-disease virus.FEBS Lett. 1996; 388: 39-42Abstract Full Text PDF PubMed Scopus (37) Google Scholar, Pilipenko et al., 2000Pilipenko E.V. Pestova T.V. Kolupaeva V.G. Khitrina E.V. Poperechnaya A.N. Agol V.I. Hellen C.U.T. A cell cycle-dependent protein serves as a template-specific translation initiation factor.Genes Dev. 2000; 14: 2028-2045PubMed Google Scholar), but for the encephalomyocarditis virus (EMCV) IRES, the PTB requirement is conditional on the nature of the reporter and the IRES variant used (Kaminski and Jackson, 1998Kaminski A. Jackson R.J. The polypyrimidine tract binding protein (PTB) requirement for internal initiation of translation of cardiovirus RNAs is conditional rather than absolute.RNA. 1998; 4: 626-638Crossref PubMed Scopus (109) Google Scholar), whereas there are conflicting reports for Theiler's murine encephalomyelitis virus (TMEV), ranging from no dependency to quite strong stimulation (Kaminski et al., 1995Kaminski A. Hunt S.L. Patton J.G. Jackson R.J. Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA.RNA. 1995; 1: 924-938PubMed Google Scholar, Pilipenko et al., 2001Pilipenko E.V. Viktorova E.G. Guest S.T. Agol V.I. Roos R.P. Cell-specific proteins regulate viral RNA translation and virus-induced disease.EMBO J. 2001; 23: 6899-6908Crossref Scopus (86) Google Scholar), which is likely to be due to the use of different strains. PTB has four RNA-binding domains (RBDs) of the RNP1/RNP2 class, although the amino acid sequences of these motifs in PTB are somewhat noncanonical. Alternative splicing results in two variants of the prototypic PTB1: PTB2 and PTB4, which differ from PTB1 by the insertion of 19 or 26 amino acids, respectively, in the linker between RBD2 and RBD3. The hierarchy of efficiency of these in an alternative splicing assay (PTB4 > PTB2 > PTB1) was almost precisely the opposite from their hierarchy in promoting initiation on the human rhinovirus IRES (Wollerton et al., 2001Wollerton M.C. Gooding C. Robinson F. Brown E.C. Jackson R.J. Smith C.W.J. Differential alternative splicing activity of isoforms of polypyrimidine tract binding protein (PTB).RNA. 2001; 7: 819-832Crossref PubMed Scopus (111) Google Scholar). PTB1 was initially considered to form dimers in solution via interactions involving the RBD2 region, but it is now known to be a monomer with a somewhat extended conformation (Monie et al., 2005Monie T.P. Hernandez H. Robinson C.V. Simpson P. Matthews S. Curry S. The polypyrimidine tract binding protein is a monomer.RNA. 2005; 11: 1803-1808Crossref PubMed Scopus (29) Google Scholar). Although the interaction of PTB with viral IRESs has been extensively studied by gel-shift and UV-crosslinking assays, and its binding sites on Type II IRESs have been mapped by footprinting (Kolupaeva et al., 1996Kolupaeva V.G. Hellen C.U.T. Shatsky I.N. Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs.RNA. 1996; 2: 1199-1212PubMed Google Scholar, Pilipenko et al., 2000Pilipenko E.V. Pestova T.V. Kolupaeva V.G. Khitrina E.V. Poperechnaya A.N. Agol V.I. Hellen C.U.T. A cell cycle-dependent protein serves as a template-specific translation initiation factor.Genes Dev. 2000; 14: 2028-2045PubMed Google Scholar, Pilipenko et al., 2001Pilipenko E.V. Viktorova E.G. Guest S.T. Agol V.I. Roos R.P. Cell-specific proteins regulate viral RNA translation and virus-induced disease.EMBO J. 2001; 23: 6899-6908Crossref Scopus (86) Google Scholar), nothing is known about the details of how it is docked onto these IRESs: the question of which RBD binds to which part of the IRES. Because it has proved impossible (so far) to address this question by X-ray crystallography or NMR spectroscopy of PTB/IRES complexes, we turned to directed hydroxyl radical probing. In this approach, an Fe(II)-EDTA moiety is attached to a strategically placed site in a given RBD, and after forming the PTB/RNA complex, hydroxyl radicals are generated at the Fe(II) by the Fenton reaction. These radicals cause RNA backbone cleavage, irrespective of structure or sequence, but because they are very short lived, cleavage is limited to sites in close proximity to the Fe(II), facilitating identification of which RNA segment is nearest that particular RBD (Culver and Noller, 2000Culver G.M. Noller H.F. Directed hydroxyl radical probing of RNA from iron(II) tethered to proteins in ribonucleoprotein complexes.Methods Enzymol. 2000; 318: 461-475Crossref PubMed Google Scholar). The EMCV IRES was chosen for these studies, because it is the best characterized IRES in terms of its secondary structure and known PTB-binding sites; thus, it provides a good platform for validating the experimental approach. PTB1 (hereafter designated simply PTB) has three Cys residues: Cys-23 in the N-terminal domain, and the other two (Cys-250, Cys-251) in RBD2 (Figure 1A). All of the mutants described here were generated in the background of the construct PTB1-1234 described by Monie et al., 2005Monie T.P. Hernandez H. Robinson C.V. Simpson P. Matthews S. Curry S. The polypyrimidine tract binding protein is a monomer.RNA. 2005; 11: 1803-1808Crossref PubMed Scopus (29) Google Scholar, renamed ΔNTD-PTB for this work: this has an N-terminal His tag and is lacking the N-terminal (amino acids 1–54) domain (Figure 1B), which has the nuclear import/export signals but plays no role in RNA binding. First, C250S and C251S mutations were made, separately and together, the latter generating a construct encoding a Cys-less protein, which was an important control used routinely in all of the probing assays. Single Cys residues were then introduced into the Cys-less background at various positions (Figure 1C), which were chosen on the basis of the published NMR structures of each RBD complexed with an oligonucleotide ligand (Oberstrass et al., 2005Oberstrass 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: 2054-2057Crossref PubMed Scopus (293) Google Scholar). The aim was to place the cysteines far enough away from the actual RNA-binding surface that the conjugated Fe-BABE moiety would be unlikely to cause steric interference with RNA binding, but in positions flanking the RNA-binding surface such that the Fe of the Fe(II)-BABE would probably be in close proximity to the bound RNA. Because the RNA-binding surface of RBD4 extends almost to the very C terminus of the protein, we chose to introduce a Cys downstream of this RBD by extending the protein with GSGC, rather than by point mutation. Each mutant was then tested for its ability to promote translation initiation on the EMCV IRES in a variant of the PTB-depleted reticulocyte lysate system described previously (Kaminski et al., 1995Kaminski A. Hunt S.L. Patton J.G. Jackson R.J. Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA.RNA. 1995; 1: 924-938PubMed Google Scholar). For these functional assays, we used a mutant EMCV IRES with an expanded 7A bulge at the three-way junction in the J-K domain (see Figure 3C), because this shows a greater response to PTB than the wild-type IRES with a 6A bulge (Kaminski and Jackson, 1998Kaminski A. Jackson R.J. The polypyrimidine tract binding protein (PTB) requirement for internal initiation of translation of cardiovirus RNAs is conditional rather than absolute.RNA. 1998; 4: 626-638Crossref PubMed Scopus (109) Google Scholar), but a wild-type (6A) IRES was used for the probing assays, except where otherwise stated. Figure 2A shows that removing the N-terminal domain and mutating C250 and C251 to serines had no effect on the stimulation of translation by PTB, and representative assays of some Cys substitution mutants are shown in Figure 2B. A minimum of two, and in some cases up to four, different mutant PTB concentrations (maximum 180 nM) were used with a constant mRNA concentration of 20 nM. In most cases, stimulation of translation was evident even at the lowest PTB concentration (22.5 nM), and it reached a maximum at 90 nM added PTB (a PTB/IRES molar ratio of 4.5). The stimulation at each concentration of mutant ΔNTD-PTB was determined relative to wild-type (FL) PTB. The averages of these relative efficiency values for each mutant are presented in Figure 2D; all retained significant activity (>70% of full-length PTB) in promoting IRES-dependent translation. Next, the single Cys mutants were conjugated with Fe(II)-BABE under standard conditions (Culver and Noller, 2000Culver G.M. Noller H.F. Directed hydroxyl radical probing of RNA from iron(II) tethered to proteins in ribonucleoprotein complexes.Methods Enzymol. 2000; 318: 461-475Crossref PubMed Google Scholar, Marzi et al., 2003Marzi S. Knight W. Brandi L. Caserta E. Soboleva N. Hill W.E. Gualerzi C.O. Lodmell J.S. Ribosomal localization of translation initiation factor IF2.RNA. 2003; 9: 958-969Crossref PubMed Scopus (45) Google Scholar). Initially, we assessed derivatization efficiency by using the fluorescent −SH reagent (see Supplemental Data, available online) described by Culver and Noller, 2000Culver G.M. Noller H.F. Directed hydroxyl radical probing of RNA from iron(II) tethered to proteins in ribonucleoprotein complexes.Methods Enzymol. 2000; 318: 461-475Crossref PubMed Google Scholar, but we noted that conjugation with Fe(II)-BABE resulted in a sufficient change of mobility on SDS-PAGE (shown for a representative set of mutants in Figure 2C) to provide a better way of assessing the extent of the reaction. Three of the 19 single Cys derivatives (R122C, C251, K402C) did not react at all with Fe-BABE; mutants Q124C, T252C, and I531GSGC were found to react partially (∼60%), but the other 13 all showed close to 100% derivatization (Figure 2D). All of the derivatized proteins retained significant activity (>60% relative to full-length, wild-type, unmodified PTB) in stimulating translation dependent on the EMCV IRES (Figure 2D). We also combined some of the mutants in pairs, with each Cys in a different RBD: (a) Q124C in RBD1 and D284C in RBD2 (“QD”); and (b) N395C in RBD3 and R491C in RBD4 (“NR”). These two were also combined to generate “QDNR” with four Cys, one in each RBD. All three of these multiple PTB mutants were functional in the in vitro translation assay (Figure 2D). The derivatization efficiency was exactly as expected from results with the two (or four) individual single Cys mutations (Figures 2C and 2D), as were the RNA cleavage sites, although fragment band intensity was slightly reduced (Figures 3A and 3B). The Fe(II)-BABE PTB derivatives, as well as the mock-conjugated Cys-less control, were preincubated at 37°C with the 5′ or 3′ end 32P-labeled IRES probe for 20 min, then ascorbic acid and hydrogen peroxide were added, and incubation continued for 30 min on ice, before the reaction was quenched by phenol extraction of the RNA fragments. Representative examples of the RNA product analysis gels with each type of end-labeled probe are shown in Figures 3A and 3B, respectively. Although the input RNA was overwhelmingly intact (full-length probe), a number of fragments appeared after addition of hydrogen peroxide and ascorbic acid, either in the absence of any protein or in the presence of the Cys-less PTB (Figure 3). The bands in the Cys-less lane were treated as the background, and bands in lanes with Fe(II)-PTB mutants were taken to be genuine products of hydroxyl radical cleavage only if they were entirely absent from the Cys-less control lane (or were present, but at decidedly lower intensity). For the first experiments, the probe encompassed nt 260–849 of the EMCV virion RNA sequence in the numbering of Duke et al., 1992Duke G.M. Hoffman M.A. Palmenberg A.C. Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation.J. Virol. 1992; 66: 1602-1609PubMed Google Scholar, representing the complete 5′UTR downstream of the polyC tract, plus the first 16 nt of the viral coding sequence. As shown in Figure 3C, this segment is conventionally regarded as consisting of nine secondary structure domains (Domains D–L, inclusive), of which Domains D–G make only a minor contribution to IRES function, whereas Domains H–L are essential (Jang and Wimmer, 1990Jang S.K. Wimmer E. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosome entry site and involvement of a cellular 57-kD RNA-binding protein.Genes Dev. 1990; 4: 1560-1572Crossref PubMed Scopus (310) Google Scholar, Duke et al., 1992Duke G.M. Hoffman M.A. Palmenberg A.C. Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation.J. Virol. 1992; 66: 1602-1609PubMed Google Scholar). With our standard RNA probe concentration of 50 nM, appreciable backbone cleavage was generally seen at equimolar Fe(II)-PTB/IRES, and a maximum signal was seen with a molar ratio of 2, although there were some exceptions that will be discussed later. For the majority of sites, raising the Fe(II)-PTB/RNA ratio much above 2-fold did not result in any increase in the intensity of the genuine cleavage product bands; rather, the background smear increased in intensity, and the relative proportion of full-length uncleaved probe decreased, especially at 8- to 10-fold molar excess (see, for example, Figures 5A–5C; Figure S2B). As this loss of full-length probe at high PTB/RNA input was much less evident with the Cys-less mutant (Figure 5A; Figure S2B), we consider it most likely to be due to nonspecific cleavages (i.e., dependent on the conjugated Fe-BABE and the consequent Fenton reaction) resulting from random interaction of the excess PTB with the RNA, rather than nuclease contamination of the PTB preparations. To estimate the relative efficiency of cleavage at any particular site, the first step was to eliminate any loading variation between lanes, by determining the intensities of a given band in the Fe(II)-PTB lane and of the equivalent region of the Cys-less control lane, and normalizing these values with respect to the corresponding full-length uncleaved probe band or (more usually) to other background bands whose intensity was found to vary between lanes in parallel with the full-length probe yield (Figures 3A and 3B). After subtracting the relevant normalized Cys-less background value, the calculated net cleavage efficiency indices for all cleavage product bands were ranked, and the ranking list was divided into three groups (representing strong, medium, and weak cleavages) following common practice (Culver and Noller, 2000Culver G.M. Noller H.F. Directed hydroxyl radical probing of RNA from iron(II) tethered to proteins in ribonucleoprotein complexes.Methods Enzymol. 2000; 318: 461-475Crossref PubMed Google Scholar). Calibrations based on a comparison of ribosome crystal structures with the cleavages generated by numerous Fe(II)-ribosomal-protein derivatives have shown that the distance between the cleavage site and the α carbon of the mutated amino acid is up to 25, 35, or 50 Å for strong, medium, and weak cleavages, respectively (Lancaster et al., 2002Lancaster L. Kiel M.C. Kaji A. Noller H.F. Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing.Cell. 2002; 111: 129-140Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). For mapping the PTB binding orientation, we focused on the strong and medium cleavages, because these will arise from high occupancy PTB binding with the RNA reasonably close to the relevant RBD residue, which will not be the case for most, if not all, of the weak cleavages. Figure 3C summarizes cleavage site data from over 35 such experiments with end-labeled IRES probes (including probes lacking Domains D–G), or more than 60 gels (because the cleavage products of many experiments were analyzed on two gels, differing in the percentage of acrylamide and/or running time), for the 9 Fe(II)-PTB derivatives that consistently gave the strongest signals. Where a given mutant produced cleavages in a number of adjacent phosphodiester bonds (as is usually the case, except for some very weak cleavages), for clarity a single arrowhead (sized according to cleavage efficiency) is used to indicate either the strongest signal or the central cleavage site in cases in which there was little difference in signal strength. The cleavage sites were highly reproducible and were initially assigned using the RNase T1 and alkaline hydrolysis ladders, with verification provided by a few analyses using reverse transcription with six different primers (data not shown), taking advantage of the higher resolution of this approach. The positions of these nine mutations in the relevant RBDs are depicted in Figure S1, which includes (in the legend) a brief summary of the cleavage patterns generated by the other seven mutants. As is clear from Figure 1C and Figure S1, our assignment of N432 to RBD3 is somewhat arbitrary, because it is actually in the middle of the short linker between RBD3 and RBD4. Because the RNA is thought to be able to loop around between these two RBDs (Oberstrass et al., 2005Oberstrass 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: 2054-2057Crossref PubMed Scopus (293) Google Scholar), N432 is close enough to give some medium cuts (though no strong cleavages), which, in some cases (e.g., in Domain E), are located between sites cut by the other RBD3 mutants (N395 and E419) and the RBD4 mutants, R491 and E518 (Figure 3C). It is immediately obvious from Figure 3C that the strong-medium cleavages generated by all mutants in a particular RBD are closely clustered in the conventional two-dimensional secondary structure map of the EMCV IRES, implying that PTB binds to the IRES in a limited number of orientations, possibly a unique orientation. Nevertheless, for each RBD, there are clearly at least two, if not three, such clusters: for both RBD1 and RBD2 in Domains F and K; RBD3 in Domains D/E, H, and L; and RBD4 in Domains E/F and G and the base of Domain I. Possible explanations for these multiple clusters are examined in the following two sections. We repeated these probing assays in the presence of the C-terminal two-thirds fragment of eIF4GI and ATP, with or without eIF4A also present because this enhances the binding of eIF4G to Domain J-K (Kolupaeva et al., 2003Kolupaeva V.G. Lomakin I.B. Pestova T.V. Hellen C.U.T. Eukaryotic initiation factors 4G and 4A mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal entry site.Mol. Cell. Biol. 2003; 23: 687-698Crossref PubMed Scopus (81) Google Scholar). No difference in the pattern of cleavages generated by the Fe(II)-PTB derivatives was seen, only a general slight decrease in signal intensity (data not shown). Probing in the presence of PTB-depleted reticulocyte lysate (i.e., under translation assay conditions) proved impossible because lysates have such high catalase activity. A striking, and somewhat surprising, feature evident in Figure 3C is the absence of cuts in Domain I (apart from the extreme base of this domain). This was confirmed in assays in which the cleavage products were analyzed by using reverse transcription from nt 754, 606, and 498 (i.e., three primers): although these clearly revealed the customary strong/medium cleavages in Domains G and H, no cleavage product bands were detected in Domain I, apart from t the bottom of this domain (data not shown). In another approach to reveal cleavages in Domain I, a 5′-end-labeled probe representing just Domains H–L (with Domains D–G deleted) was used, but although the majority of the cleavages in the 3′ side of Domain H were visible at the bottom of the gel, as was the strong cleavage given by D284C in Domain K at the top, no fragments resulting from cutting in Domain I were seen in the middle of the gel (Figure 4A). We went on to make two deletions removing most of Domain I, but in both cases leaving a short stem-loop in an attempt to maintain the same spatial relationship between the upstream Domains D–H and the downstream Domains J–L as in the full-length IRES. These deletions made no difference to the cleavages in Domains D–H detected with the 5′-end-labeled probe (Figure 4B), nor those in Domains J–L seen with the 3′-end-labeled probe (Figure 4C). Moreover, in the latter case, many of the strong cleavages seen in Domains E–H were also clearly visible (Figure 4C). We conclude that there are no close contacts between any of the four RBDs of PTB and the major part of Domain I, and that deletion of the majority of Domain I does not appear to significantly perturb the interaction of PTB with the remainder of the IRES, suggesting that Domain I may be relatively independent and spatially separate from the other IRES domains. There are three possible explanations for why mutants in any one RBD cause cleavage at two or more widely separated sites (Figure 3C): (1) the IRES may bind two (or more) PTBs simultaneously; (2) the IRES may bind only one PTB, but in two (or more) alternative orientations; or (3) the IRES may be binding only a single PTB in a unique orientation, but the three-dimensional tertiary structure of the IRES brings into close proximity the (apparently) distant clusters of cleavage sites specific for any one RBD. In an attempt to distinguish between these possibilities, we examined the influence of PTB concentration on the cleavage signals produced by two selected mutants in RBD2 (N219C and D284C), which generate strong or medium cleavages in both Domain F and Domain K (Figure 3C). In order to allow the cleavages in both domains to be examined in a single reaction and in a single gel track, the probe was end labeled at both ends, with markers provided by using probes labeled uniquely at either end. With both derivatives, the cleavage signals in Domain F were already nearly maximal at 40 nM Fe(II)-PTB, whereas the signals in Domain K were only detectable at the higher concentration of 100 nM PTB (Figures 5A and 5B), implying that RBD2 binds with higher affinity to Domain F than to Domain K. In addition, hydroxyl radical probing of a 5′-end-labeled RNA with increasing amounts of E419C showed that cleavages produced in Domain D were more than half-maximal at 50 nM, whereas those in Domain H started appearing only at 100 nM (Figure 5C), implying a higher-affinity interaction of RBD3 with Domain D than Domain H. All of these observations suggest that the full-length IRES can bind two PTBs simultaneously (but with different affinities), although binding a single PTB in two alternative orientations is not absolutely ruled out. Because the EMCV IRES is too large to give band shifts sharp enough to unambiguously determine the stoichiometry of PTB/IRES interactions by EMSAs, we turned to the much more precise method of mass spectrometry. For this, PTB/IRES complexes were initially assembled in 58 mM ammonium acetate (pH 7.4), 2 mM Mg2+, with RNA at 10 μM, followed by buffer exchange into 100 mM ammonium acetate (except in the case of the ΔD–I probe, where the PTB/RNA complexes dissociated under the conditions of reduced Mg2+ and raised NH4+) with 2-fold dilution. Wild-type ΔNTD-PTB was used for these experiments at a nominal input molar ratio of 0.5:1, 1:1, or 2:1 with respect to IRES RNA. In view of the fact that the total RNA concentration (after dilution) was 250-fold greater than in the in vitro translation assays (or 100-fold greater than for directed hydroxyl radical probing), and given that estimates of the KD of the interaction between PTB and various Type II picornavirus IRESs all fall in the 20–50 nM range (Song et al., 2005Song Y. Tzima E. Ochs K. Bassili G. Trusheim H. Linder M. Preissner K.T. Niepmann M. Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation.RNA. 2005; 11: 1809-1824Crossref PubMed Scopus (70) Google Scholar), we consider that the nominal 1:1 PTB/RNA input ratio will approximate most closely to the degree of saturation of the RNA with bound PTB pertaining in the translation and probing assays. The results with four different IRES derivatives (each with 16 nt viral coding sequence at the 3′-end) fall into two classes. The ΔD–G IRES and the more severely" @default.
• W2076580369 created "2016-06-24" @default.
• W2076580369 creator A5001267625 @default.
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• W2076580369 date "2009-06-01" @default.
• W2076580369 modified "2023-10-15" @default.
• W2076580369 title "Polypyrimidine Tract Binding Protein Stabilizes the Encephalomyocarditis Virus IRES Structure via Binding Multiple Sites in a Unique Orientation" @default.
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• W2076580369 doi "https://doi.org/10.1016/j.molcel.2009.04.015" @default.
• W2076580369 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19524536" @default.
• W2076580369 hasPublicationYear "2009" @default.
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