A protein of molecular size 62,000 daltons (p62) was detected in HeLa cell nuclear extracts by UV cross-linking to mRNA precursors. p62 binds specifically to the polypyrimidine tract of the 3' splice site region of introns. p62 purified to homogeneity binds the polypyrimidine tract of pre-mRNAs. This binding does not require the AG dinucleotide at the 3' splice site. Alterations in the polypyrimidine tract that reduce the binding of p62 yield a corresponding reduction in the efficiency of formation of a U2 snRNP/pre-mRNA complex and splicing. The p62 protein is retained in the spliceosome, where it remains bound to the pre-mRNA. This polypyrimidine tract binding protein (pPTB) is proposed to be a critical component in recognition of the 3' splice site during splicing (Garcia-Blanco, 1989).
Studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7. Factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage have been identified using an RNA mobility-shift assay. A protein that exhibits these sequence specific RNA binding properties has been purified to apparent homogeneity. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: (1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and (2) the intron regulatory element that is involved in the repression of exon 7. These results demonstrate that the sequence requirements for PTB binding are different from those previously reported and show that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites (Mulligan, 1992).
Polypyrimidine tract binding protein (PTB) is a 57 kD hnRNP protein (hnRNP I) that binds to the pyrimidine tract typically found near the 3' end of introns. Primary sequence analysis suggests that PTB contains four RNA recognition motifs (RRMs). Data from comparative structural and deletional analysis of PTB are consistent with the presence of a four reiterated domain structure. Since PTB exists in solution as a homodimer, it contains an oligomeric array of eight RRMs. Though the function of RRMs in a monomeric context has been addressed, the significance of their presence in an oligomeric context has not been investigated. To correlate structural motifs with function, the RNA binding properties have been analyzed of wild-type and deletion constructs of PTB that contain RRMs in both an oligomeric and monomeric context. These studies indicate that there is not a strong correlation between the RNA binding affinity and specificity upon oligomerization. However, the mode of RNA interaction and dimerization is linked. The primary contributor to the free energy of PTB binding and the primary determinant for RNA binding specificity resides in RRM 3, while the primary contributor to dimer stabilization coincides with residues in RRM 2 (Perez, 1997).
Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, the binding has been characterized of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. A new 60-kDa tissue-specific protein has been identified that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins (Markovtsov, 2000).
The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays (Liu, 2002).
Polypyrimidine tract-binding protein (PTB) is an abundant widespread RNA-binding protein with roles in regulation of pre-mRNA alternative splicing and 3'-end processing, internal ribosomal entry site-driven translation, and mRNA localization. Tissue-restricted paralogs of PTB have been reported in neuronal and hematopoietic cells. These proteins are thought to replace many general functions of PTB, but to have some distinct activities, e.g. in the tissue-specific regulation of some alternative splicing events. A fourth rodent PTB paralog (smPTB) has been identified and characterized that is expressed at high levels in a number of smooth muscle tissues. Recombinant smPTB localizes to the nucleus, binds to RNA, and is able to regulate alternative splicing. It is suggested that replacement of PTB by smPTB might be important in controlling some pre-mRNA alternative splicing events (Gooding, 2003).
The perinucleolar compartment (PNC) is a nuclear substructure present in transformed cells. The PNC is defined by high concentrations of certain RNA binding proteins and a subset of small RNAs transcribed by RNA polymerase III (pol III), including the signal recognition particle RNA and an Alu RNA as reported in this study. To determine if the PNC is dependent on pol III transcription, HeLa cells were microinjected with the selective pol III inhibitor, Tagetin. This resulted in disassembly of the PNC, whereas inhibition of pol I by cycloheximide or pol II by alpha-amanitin did not significantly affect the PNC. However, overexpression of one of the PNC-associated RNAs from a pol II promoter followed by injection of Tagetin blocked the Tagetin-induced PNC disassembly, demonstrating that it is the RNA rather than pol III activity that is important for the PNC integrity. To elucidate the role of the PNC-associated protein PTB, its synthesis was inhibited by siRNA. This resulted in a reduction of the number of PNC-containing cells and the PNC size. Together, these findings suggest, as a working model, that PNCs may be involved in the metabolism of specific pol III transcripts in the transformed state and that PTB is one of the key elements mediating this process (Wang, 2003).
The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remains intact and is N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggest differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provide evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociates from PTB and binds new partners, including the U1--70K and SR proteins and therefore may acquire new functions (Shav-Tal, 2001).
Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induces very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor (Gromak, 2003).
Cytoplasmic localization of mRNA molecules is a powerful mechanism for generating cell polarity. In vertebrates, one paradigm is localization of Vg1 RNA within the Xenopus oocyte, a process directed by recognition of a localization element within the Vg1 3' UTR. Specific base changes within the localization element abolish both localization in vivo and binding in vitro by a single protein, VgRBP60. VgRBP60 is homologous to a human hnRNP protein, hnRNP I, and combined immunolocalization and in situ hybridization demonstrate striking colocalization of hnRNP I and Vg1 RNA within the vegetal cytoplasm of the Xenopus oocyte. These results implicate a novel role in cytoplasmic RNA transport for this family of nuclear RNA-binding proteins (Cote, 1999).
Mutually exclusive use of exons IIIb or IIIc in FGF-R2 transcripts requires the silencing of exon IIIb. This repression is mediated by silencer elements upstream and downstream of the exon. Both silencers bind the polypyrimidine tract binding protein (PTB) and PTB binding sites within these elements are required for efficient silencing of exon IIIb. Recruitment of MS2-PTB fusion proteins upstream or downstream of exon IIIb causes repression of this exon. Depletion of endogenous PTB using RNAi increases exon IIIb inclusion in transcripts derived from minigenes and from the endogenous FGF-R2 gene. These data demonstrate that PTB is a negative regulator of exon definition in vivo (Wagner, 2002).
Deletion of PTB binding sites and knockdown of PTB both leads to an approximately 3-fold increase in exon IIIb inclusion, whereas deletion of a complete intronic control element leads to a 10-fold increase in exon inclusion. The most likely explanation for these results is that PTB collaborates with other unidentified factors, which bind to the 5' control element. The need for multiple factors to bind adjacent elements to integrate an alternative splicing outcome has been noted in several cases. HnRNP H, hnRNP F, KSRP, and nPTB have been found to bind the downstream splicing enhancer, which is required for inclusion of the N1 in c-src mRNAs in neural tissues. The tissue-specific inclusion of the alternative exon 5 of cardiac troponin-T appears to require the integrated activity of PTB and members of the msl family of factors. PTB associates with FBP and Sam68 on the intron upstream of the regulated exon 7 in rat ß-tropomyosin transcripts. The need to regulate a vast number of alternative splicing events has been solved by the integration of the activity of a limited number of factors that by combinatorial assortment can lead to very large number of functional states. An elegant binary switch provided by a single alternative splicing factor, as is the case for Sxl protein in D. melanogaster, may be reserved for crucial decisions, such as sex determination, which are made very early in development (Wagner, 2002).
It is clear that the factors that mediate silencing of exon IIIb via the upstream intronic splicing silencer and the intronic control element are present and active in both fibroblasts and epithelial cells. How then is exon IIIb included in FGF-R2 mRNAs in epithelial cells? It is likely that a cell type-specific factor or perhaps a combination of factors results in the specific derepression of exon IIIb. Exon IIIb activating factors are recruited via the intronic activating sequence 2 and the upstream activating element ISAR; these sites work in concert and form a secondary structure that is required for their function. Given that intronic activating sequence 2 is embedded within intronic element, it is reasonable to predict that the IAS2-ISAR structure will disrupt the silencing topology. Thus exon IIIb inclusion in epithelial cells is likely achieved by countering the repression mechanism instituted by PTB and other yet nidentified splicing repressors (Wagner, 2002).
Pancreatic beta-cells store insulin in secretory granules that undergo exocytosis upon glucose stimulation. Sustained stimulation depletes beta-cells of their granule pool, which must be quickly restored. However, the factors promoting rapid granule biogenesis are unknown. Beta-cell stimulation induces the nucleocytoplasmic translocation of polypyrimidine tract-binding protein (PTB). Activated cytosolic PTB binds and stabilizes mRNAs encoding proteins of secretory granules, thus increasing their translation, whereas knockdown of PTB expression by RNA interference (RNAi) results in the depletion of secretory granules. These findings may provide insight for the understanding and treatment of diabetes, in which insulin secretion is typically impaired (Knoch, 2004).
PTB functions as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are capable of mediating developmental splicing control. Altered expression of PTB isoforms during cerebellar development, as documented by Western blot analysis, is proposed to be a contributing mechanism (Zhang, 1999).
The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. The nucleo-cytoplasmic transport of PTB is regulated by the 3',5'-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB (Xie, 2003).
PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3'-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5' splice site sequence to form a pseudoexon. The binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB is antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminates enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation (Lou, 1999).
Stabilization of insulin mRNA in response to glucose is a significant component of insulin production, but the mechanisms governing this process are unknown. Insulin mRNA is a highly abundant messenger and the content of this mRNA is mainly controlled by changes in messenger stability. Specific binding of the polypyrimidine tract-binding protein to a pyrimidine-rich sequence located in the 3'-untranslated region (3'-UTR) of insulin mRNA has been demonstrated. This binding is increased in vitro by dithiothreitol and in vivo by glucose. Inhibition of polypyrimidine tract-binding protein binding to the pyrimidine-rich sequence by mutation of the core binding site results in a destabilization of a reporter gene mRNA. Thus, glucose-induced binding of polypyrimidine tract-binding protein to the 3'-UTR of insulin mRNA could be a necessary event in the control of insulin mRNA levels (Tillmar, 2002).
Polypyrimdine tract binding protein (PTB) is a regulator of alternative splicing, mRNA 3' end formation, mRNA stability and localization, and IRES-mediated translation. Transient overexpression of PTB can influence alternative splicing, sometimes resulting in nonphysiological splicing patterns. Alternative skipping of PTB exon 11 leads to an mRNA that is removed by nonsense-mediated decay; this pathway consumes at least 20% of the PTB mRNA in HeLa cells. Exon 11 skipping is itself promoted by PTB in a negative feedback loop. This autoregulation may serve both to prevent disruptively high levels of PTB expression and to restore nuclear levels when PTB is mobilized to the cytoplasm. These findings suggest that alternative splicing can act not only to generate protein isoform diversity but also to quantitatively control gene expression and complement recent bioinformatic analyses, indicating a high prevalence of human alternative splicing leading to nonsense-mediated decay (Wollerton, 2004).
The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. Conserved intronic elements flanking N1 have been identified that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. These upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds (Chan, 1997).
The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon uses a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression can be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts can be activated by (1) truncation of the transcript between the SM polypyrimidine tract and exon, (2) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha-tropomyosin (TM), and (3) depletion of the splicing extract by using biotinylated alpha-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB is the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlates with their ability to bind PTB. Furthermore, repression of alpha-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA can be specifically restored by the addition of recombinant PTB. Thus, alpha-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB (Southby, 1999).
Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) transcripts involves the mutually exclusive usage of exons IIIb and IIIc to produce two different receptor isoforms. Appropriate splicing of exon IIIb in rat prostate cancer DT3 cells requires a previously described cis element (ISAR, for intronic splicing activator and repressor) which represses the splicing of exon IIIc and activates the splicing of exon IIIb. This element is nonfunctional in rat prostate AT3 cells, which repress exon IIIb inclusion and splice to exon IIIc. An intronic element upstream of exon IIIb has been identified that causes repression of exon IIIb splicing. Deletion of this element abrogates the requirement for ISAR in order for exon IIIb to be spliced in DT3 cells and causes inappropriate inclusion of exon IIIb in AT3 cells. This element consists of two intronic splicing silencer (ISS) sequences, ISS1 and ISS2. The ISS1 sequence is pyrimidine rich, and in vitro cross-linking studies demonstrate binding of polypyrimidine tract binding protein (PTB) to this element. Competition studies demonstrate that mutations within ISS1 that abolish PTB binding in vitro alleviate splicing repression in vivo. Cotransfection of a PTB-1 expression vector with a minigene containing exon IIIb and the intronic splicing silencer element demonstrate PTB-mediated repression of exon IIIb splicing. Furthermore, all described PTB isoforms are equally capable of mediating this effect. These results support a model of splicing regulation in which exon IIIc splicing does not represent a default splicing pathway but rather one in which active repression of exon IIIb splicing occurs in both cells and in which DT3 cells are able to overcome this repression in order to splice exon IIIb (Carstens, 2000).
The role of polypyrimidine tract binding protein in repressing splicing of the c-src neuron-specific N1 exon was investigated. Immunodepletion/add-back experiments demonstrate that PTB is essential for splicing repression in HeLa extract. When splicing is repressed, PTB cross-links to intronic CUCUCU elements flanking the N1 exon. Mutation of the downstream CU elements causes dissociation of PTB from the intact upstream CU elements and allows splicing. Thus, PTB molecules bound to multiple elements cooperate to repress splicing. Interestingly, in neuronal WERI-1 cell extract where N1 is spliced, PTB also binds to the upstream CU elements but is dissociated in the presence of ATP. It is concluded that splicing repression by PTB is modulated in different cells by a combination of cooperative binding and ATP-dependent dissociation (Chou, 2000).
Inclusion of cardiac troponin T (cTNT) exon 5 in embryonic muscle requires conserved flanking intronic elements (MSEs). ETR-3, a member of the CELF family, binds U/G motifs in two MSEs and directly activates exon inclusion in vitro. Binding and activation by ETR-3 are directly antagonized by polypyrimidine tract binding protein (PTB). Dominant-negative mutants have been used to demonstrate that endogenous CELF and PTB activities are required for MSE-dependent activation and repression in muscle and nonmuscle cells, respectively. Combined use of CELF and PTB dominant-negative mutants provides an in vivo demonstration that antagonistic splicing activities exist within the same cells. It is concluded that cell-specific regulation results from the dominance of one state among actively competing regulatory states, rather than modulation of a nonregulated default state (Charlet-B., 2002).
The Xenopus alphafast-tropomyosin gene contains at its 3' end a composite internal/3' terminal exon (exon 9A9') which is subjected to three different patterns of splicing according to the cell type. Exon 9A9' is included as a terminal exon in the myotome and as an internal exon in adult striated muscles whereas it is skipped in non-muscle cells. An in vivo model has been developed based on transient expression of minigenes encompassing the regulated exon 9A9' in Xenopus oocytes and embryos. The different alpha-tropomyosin minigenes recapitulate the splicing pattern of the endogenous gene and valuable tools have been constituted to seek regulatory sequences involved in exon 9A9' usage. A mutational analysis led to the identification of an intronic element that is involved in the repression of exon 9A9' in non-muscle cells. This element harbors four polypyrimidine track-binding protein (PTB) binding sites that are essential for the repression of exon 9A9'. Using UV cross-linking and immuno-precipitation experiments it has been shown that XPTB interacts with these PTB binding sites. Finally, it is shown that depletion of endogenous XPTB in Xenopus embryos using a morpholino based translational inhibition strategy results in exon 9A9' inclusion in embryonic epidermal cells. These results demonstrate that XPTB is required in vivo to repress the terminal exon 9A9' and suggest that PTB could be a major actor in the repression of regulated 3' terminal exon (Hamon, 2004).
Polypyrimidine tract binding protein 1 (PTB: Drosophila homolog Hephaestus) binds and activates the Apaf-1 internal ribosome entry segment (IRES) when the protein upstream of N-ras (unr; a single-stranded RNA binding protein which contains five cold shock domains, Drosophila homolog CG7015) is prebound. The Apaf-1 IRES is highly active in neuronal-derived cell lines due to the presence of the neuronal-enhanced version of PTB, nPTB. The unr and PTB/nPTB binding sites have been located on the Apaf-1 IRES RNA, and a structural model for the IRES bound to these proteins has been derived. The ribosome landing site has been located to a single-stranded region, and this is generated by the binding of the nPTB and unr to the RNA. These data suggest that unr and nPTB act as RNA chaperones by changing the structure of the IRES into one that permits translation initiation (Mitchell, 2003).
The regulatory mechanisms controlling cell death are complex, and in addition to control of transcription, the expression of proteins that are involved in apoptosis is regulated by control of translation. Indeed, many mRNAs whose protein products are involved in apoptosis are translated by the alternative mechanism of internal ribosome entry. This process is mediated by a complex RNA structural element located in the 5' untranslated region (UTR) of the mRNA termed an internal ribosome entry segment (IRES). During apoptosis cap-dependent translation initiation is very much reduced, yet expression of certain key proteins required for this process is maintained by internal ribosome entry. Thus c-myc, DAP5, and XIAP IRESes function to maintain expression of these proteins following apoptosis. Apaf-1 translation is solely initiated by internal ribosome entry, but to date the only situation where a small increase in Apaf-1 IRES function has been observed is following genotoxic stress. Given the importance of Apaf-1 during brain development, it is possible that the Apaf-1 IRES is required for expression of this protein in the developing brain. In this regard the FGF-2 IRES has been shown to be active in adult brain while in developing embryos both the FGF-2 and c-myc IRESes are active. This suggests that certain IRES trans-acting factors (ITAFs) are not present in the fully differentiated cell types, and these and additional studies have demonstrated that the function of certain cellular IRESes varies considerably with cell type. Most cellular IRESes are inactive in vitro, again suggesting an absolute requirement for ITAFs that are not present in these systems. However, very few ITAFs have been identified for cellular IRESes although the auto-antigen La has been shown to interact with the XIAP IRES and hnRNPC has been shown to interact with the PDGF IRES. The Apaf-1 IRES requires both polypyrimidine tract binding protein (PTB; a protein that has a role in regulating splicing as well as aiding internal ribosome entry of certain viral IRESes and upstream of N-ras). PTB only binds to the Apaf-1 IRES RNA if unr is prebound suggesting that unr is required to attain the correct structural conformation of the Apaf-1 IRES (Mitchell, 2003).
HMGA2 is an architectural nuclear factor that plays an important role in development and tumorigenesis, but mechanisms regulating its expression are largely unknown. The proximal promoters of the mouse and human genes coding for HMGA2 contain a conserved polypyrimidine/polypurine (ppyr/ppur) element which constitutes a multiple binding site for Sp1 and Sp3 transcription factors. This region can adopt a single-stranded DNA conformation, as demonstrated in vitro by S1 nuclease sensitivity on supercoiled plasmids, indicative of an intramolecular triple-helical H-DNA structure. Moreover, PTB (polypyrimidine tract binding protein), a member of the hnRNP family, binds the pyrimidine strand of Hmga2 as well as similar ppyr/ppur elements of the c-Ki-ras (R.Y) and c-myc P1 promoters. Transfection experiments indicate that non-B-DNA conformers of the ppyr/ppur tract of the Hmga2 promoter contribute to positive transcriptional activity. A transcriptional mechanism is proposed, one acting on the Hmga2 non-B-DNA structure and functioning through interconversion between double-stranded and single-stranded DNA. Such a mechanism seems to be adopted by an increasing number of genes, mainly growth-related (Rustighi, 2002).
Initiation of translation of the animal picornavirus RNAs is via a mechanism of direct internal ribosome entry, which requires a substantial segment of the viral 5'-untranslated region, generally known as the IRES (for internal ribosome entry site). Because, however, translation of the RNAs of members of the enterovirus, and more especially, the rhinovirus subgroups of the Picornaviridae is restricted in the reticulocyte lysate system, but is greatly stimulated by the addition of HeLa cell extracts, the implication is that, in these cases, internal initiation also requires cellular trans-acting factors that are more abundant in HeLa cell extracts than in rabbit reticulocytes. This assay was used as the basis of a functional assay for the purification of the HeLa cell factors required for translation dependent on the human rhinovirus-2 (HRV) IRES. There are two such HeLa cell factors separable by ion-exchange chromatography, each of which is individually active in the assay, although their combined effect is synergistic. One of these activities is shown to be polypyrimidine-tract binding protein (PTB) on the grounds that (1) the activity copurifies to homogeneity with PTB and (2) recombinant PTB expressed in Escherichia coli stimulates HRV IRES-dependent translation with a specific activity similar to that of the purified HeLa cell factor. Furthermore, it is shown that recombinant PTB also stimulates the translation of RNAs bearing the poliovirus type 1 (Mahoney) IRES (Hunt, 1999).
Bip is a chaperone protein that can also regulate the unfolded protein response of the cell. Translation initiation of human Bip mRNA is directed by an internal ribosomal entry site (IRES) located in the 5' non-translated region. As of yet, no trans-acting factor possibly involved in this process has been identified. For the encephalomyocarditis virus and other picornaviruses, polypyrimidine tract-binding protein (PTB) has been found to enhance the translation through IRES elements, probably by interaction with the IRES structure. PTB specifically binds to the central region (nt 50-117) of the Bip 5' non-translated region. Addition of purified PTB to rabbit reticulocyte lysate and overexpression of PTB in Cos-7 cells selectively inhibit Bip IRES-dependent translation. However, depletion of endogenous PTB or addition of an RNA interacting with PTB enhanced the translational initiation directed by Bip IRES. These results suggest that PTB can either enhance or inhibit IRES-dependent translation depending on mRNAs (Kim, 2000).
Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) and requires both eukaryotic initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). The requirements for trans-acting factors differ between related picornavirus IRESs and can account for cell type-specific differences in IRES function. The neurovirulence of Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) is completely attenuated by substituting its IRES with that of foot-and-mouth disease virus (FMDV). Reconstitution of initiation using fully fractionated translation components indicates that 48S complex formation on both IRESs requires eIF2, eIF3, eIF4A, eIF4B, eIF4F, and the pyrimidine tract-binding protein (PTB) but that the FMDV IRES additionally requires ITAF(45), also known as murine proliferation-associated protein (Mpp1), a proliferation-dependent protein that is not expressed in murine brain cells. ITAF(45) does not influence assembly of 48S complexes on the TMEV IRES. Specific binding sites for ITAF(45), PTB, and a complex of the eIF4G and eIF4A subunits of eIF4F map onto the FMDV IRES, and the cooperative function of PTB and ITAF(45) in promoting stable binding of eIF4G/4A to the IRES is characterized by chemical and enzymatic footprinting. The data indicate that PTB and ITAF(45) act as RNA chaperones that control the functional state of a particular IRES; their cell-specific distribution may constitute a basis for cell-specific translational control of certain mRNAs (Pilipenko, 2000).
The polypyrimidine tract-binding protein (PTB) is a nuclear protein that regulates alternative splicing. In addition, it plays a role in the cytoplasm during infection by some viruses and functions as a positive effector of hepatitis B virus RNA export. Thus, it presumably contains a nuclear export signal (NES). Using a heterokaryon export assay in transfected cultured cells, it has been shown that the N-terminal 25 amino acid residues of PTB function as an autonomous NES, with residues 11-16 being important for NES activity. Unlike the heteronuclear ribonucleoprotein A1 NES, this NES is separable from the nuclear localization signal, which spans the entire N-terminal 60 residues of PTB. The PTB NES cannot be shown to bind to CAS or Crm1, cellular receptors known to export proteins from the nucleus, and it functions in the presence of leptomycin B, a specific inhibitor of Crm1-dependent export. PTB deleted of its NES, unlike wild type PTB, does not stimulate the export of hepatitis B virus RNA. Therefore, the PTB NES is a functionally important domain of this multifunctional protein that utilizes an unknown export receptor (Li, 2002).
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