Exon repression by polypyrimidine tract binding protein

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3' splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex.     

The polypyrimidine tract binding protein is a monomer

The polypyrimidine tract binding (PTB) protein is a potent regulator of alternative mRNA splicing. It also participates in other essential cellular functions, including translation initiation and polyadenylation. Several published reports have suggested that the protein forms a dimer in solution, a feature that has been widely incorporated into mechanistic models of protein function. However, recent studies have provided indications that full-length PTB is a monomer. Here we present new biophysical and biochemical evidence supporting the monomeric status of the protein. By use of blue-native polyacrylamide gel electrophoresis and size-exclusion chromatography, PTB was observed as a single molecular species under native reducing environments, though in oxidizing conditions, a larger protein species was also detected. Further analyses of wild-type and mutant PTB molecules with SDS-PAGE and time-of-flight electrospray ionization mass spectroscopy confirmed these observations. They also identified the single reduced species as monomeric PTB and the higher-molecular-weight nonreduced species as disulphide-linked PTB dimer mediated by Cys23. Our results indicate that the use of oxidizing environments in previous studies is likely to have contributed to the mis-assignment of PTB as a dimer. Although purified PTB may form disulphide-linked dimers under these conditions, in the reducing intracellular environment the protein will be monomeric. These findings have implications for the construction of models of PTB function in regulating mRNA metabolism.     

Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon.

Splicing of exons 2 and 3 of a-tropomyosin (TM) involves mutually exclusive selection of either exon 3, which occurs in most cells, or of exon 2 in smooth muscle (SM) cells. The SM-specific selection of exon 2 results from the inhibition of exon 3. At least two essential cis-acting elements are required for exon 3 inhibition, the upstream and downstream regulatory elements (URE and DRE). These elements are essential for repression of TM exon 3 in SM cells, and also mediate a low level of repression of exon 3 in an in vitro 5' splice site competition assay in HeLa extracts. Here, we show that the DRE consists of at least two discrete components, a short region containing a number of UGC motifs, and an essential pyrimidine-rich tract (DY). We show that the specific sequence of the DY element is important and that DY is able to bind to factors in HeLa nuclear extracts that mediate a low background level of exon 3 skipping. Deletion of a sequence within DY identified as an optimal binding site for PTB impairs (1) regulation of splicing in vivo, (2) skipping of exon 3 in an in vitro 5' splice site competition, (3) the ability of DY competitors to affect the 5' splice site competition in vitro, and (4) binding of PTB to DY. Addition of recombinant PTB to in vitro splicing reactions is able to partially reverse the effects of the DY competitor RNA. The data are consistent with a model for regulation of TM splicing that involves the participation of both tissue-specific and general inhibitory factors and in which PTB plays a role in repressing both splice sites of exon 3.     

Characterization of the internal IRES element of the zebrafish connexin55.5 reveals functional implication of the polypyrimidine tract binding protein

Background  

Connexin55.5 (Cx55.5) is a gap junction protein with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the receptor-horizontal cell complex in form of hemichannels where light-dependent plasticity occurs. This connexin is the first example of a gap junction protein processed to form two protein isoforms from a monocistronic message by an IRES mediated process. The nuclear occurrence of a carboxy-terminal fragment of this protein provides evidence that this gap junction protein may participate in a putative cytoplasmic to nuclear signal transfer.     

Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA.

The requirement of PTB, polypyrimidine tract binding protein, for internal initiation of translation has been tested using an RNA affinity column to deplete rabbit reticulocyte lysates of PTB. The affinity column was prepared by coupling CNBr-activated Sepharose with the segment of the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA previously shown to bind PTB. Lysates passed through this column were devoid of PTB, and were incapable of internal initiation of translation dependent on the EMCV 5'-untranslated region, while retaining the capacity for translation dependent on ribosome scanning. Full activity for internal initiation was restored by the addition of recombinant PTB at the physiologically relevant concentration of about 5 micrograms/mL. When various PTB deletion mutants were tested, it was found that this activity required virtually the full-length protein. Thus, PTB is an essential protein for internal initiation promoted by the EMCV 5'-untranslated region. However, the PTB-depleted lysate retained the capacity for internal initiation promoted by the 5'-untranslated regions of another cardiovirus, Theiler's murine encephalomyelitis virus, and of the unrelated hepatitis C virus, and in neither case did addition of recombinant PTB stimulate internal initiation. Therefore, PTB is not a universal internal initiation factor that is indispensable in every case of internal ribosome entry.     

Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein

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, we characterized the binding 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. We also identified a new 60-kDa tissue-specific protein 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.     

The polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation

Mammalian host factors required for efficient viral gene expression and propagation have been often recalcitrant to genetic analysis. A case in point is the function of cellular factors that trans-activate internal ribosomal entry site (IRES)-driven translation, which is operative in many positive-stranded RNA viruses, including all picornaviruses. These IRES trans-acting factors have been elegantly studied in vitro, but their in vivo importance for viral gene expression and propagation has not been widely confirmed experimentally. Here we use RNA interference to deplete mammalian cells of one such factor, the polypyrimidine tract binding protein, and test its requirement in picornavirus gene expression and propagation. Depletion of the polypyrimidine tract binding protein resulted in a marked delay of particle propagation and significantly decreased synthesis and accumulation of viral proteins of poliovirus and encephalomyocarditis virus. These effects could be partially restored by expression of an RNA interference-resistant exogenous polypyrimidine tract binding protein. These data indicate a critical role for the polypyrimidine tract binding protein in picornavirus gene expression and strongly suggest a requirement for efficient IRES-dependent translation.     

NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element

The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.     

Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES

The cellular protein, poly(rC) binding protein 2 (PCBP2), is known to function in picornavirus cap-independent translation. We have further examined the RNA binding properties and protein-protein interactions of PCBP2 necessary for translation. We have studied its putative multimerization properties utilizing the yeast two-hybrid assay and in vitro biochemical methods, including glutathione S-transferase (GST) pull-down assays and gel filtration. Through genetic analysis, the multimerization domain has been localized to the second K-homologous (KH) RNA binding domain of the protein between amino acids 125 and 158. To examine the function of multimerization in poliovirus translation, we utilized the truncated protein, DeltaKH1-PCBP2, which is capable of multimer formation, but does not bind poliovirus stem-loop IV RNA (an interaction required for translation). Utilizing RNA binding and in vitro translation assays, this protein was shown to act as a dominant negative, suggesting that PCBP2 multimerization functions in poliovirus translation and RNA binding. Additionally, PCBP2 containing a deletion in the multimerization domain (DeltaKH2-PCBP2) was not able to bind poliovirus stem-loop IV RNA and could not rescue translation in extracts that were depleted of endogenous PCBP2. Results from these experiments suggest that the multimerization of PCBP2 is required for efficient RNA binding and cap-independent translation of poliovirus RNA. By examining the functional interactions of the cellular protein PCBP2, we have discovered a novel determinant in the mechanism of picornavirus cap-independent translation.     

Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay

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. Here, we show that alternative skipping of PTB exon 11 leads to an mRNA that is removed by NMD and that this pathway consumes at least 20% of the PTB mRNA in HeLa cells. We also show that 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. Our 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 NMD.     

The polypyrimidine tract binding protein (PTB) requirement for internal initiation of translation of cardiovirus RNAs is conditional rather than absolute.

Picornavirus RNAs are translated by an unusual mechanism of internal ribosome entry that requires a substantial segment of the viral 5'-untranslated region, generally known as the internal ribosome entry segment (IRES), and in some circumstances may require cellular trans-acting proteins, particularly polypyrimidine tract binding protein (PTB). It is shown here that for encephalomyocarditis virus (EMCV), the PTB dependence of IRES function in vitro is determined partly by the nature of the reporter cistron, and more especially by the size of an A-rich bulge in the IRES. With a wild-type EMCV IRES (which has a bulge of 6 As), translation is effectively independent of PTB provided the IRES is driving the synthesis of EMCV viral polyprotein. With an enlarged (7A) bulge and heterologous reporters, translation is highly dependent on PTB. Intermediate levels of PTB dependence are seen with a 7A bulge IRES driving viral polyprotein synthesis or a wild-type (6A) bulge IRES linked to a heterologous reporter. None of these parameters influenced the binding of PTB to the high-affinity site in the IRES. These results argue that PTB is not an essential and universal internal initiation factor, but, rather, that when it is required, its binding to the IRES helps to maintain the appropriate higher-order structure and to reverse distortions caused, for example, by an enlarged A-rich bulge.     

Hephaestus encodes a polypyrimidine tract binding protein that regulates Notch signalling during wing development in Drosophila melanogaster

We describe the role of the Drosophila melanogaster hephaestus gene in wing development. We have identified several hephaestus mutations that map to a gene encoding a predicted RNA-binding protein highly related to human polypyrimidine tract binding protein and Xenopus laevis 60 kDa Vg1 mRNA-binding protein. Polypyrimidine tract binding proteins play diverse roles in RNA processing including the subcellular localization of mRNAs, translational control, internal ribosome entry site use, and the regulation of alternate exon selection. The analysis of gene expression in imaginal discs and adult cuticle of genetic mosaic animals supports a role for hephaestus in Notch signalling. Somatic clones lacking hephaestus express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of DELTA: These results lead to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta. This is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in the Notch signalling pathway.     

Antagonistic regulation of alpha-actinin alternative splicing by CELF proteins and polypyrimidine tract binding protein

The alpha-actinin gene has a pair of alternatively spliced exons. The smooth muscle (SM) exon is repressed in most cell types by polypyrimidine tract binding protein (PTB). CELF (CUG-BP and ETR3-like factors) family proteins, splicing regulators whose activities are altered in myotonic dystrophy, were found to coordinately regulate selection of the two alpha-actinin exons. CUG-BP and ETR3 activated the SM exon, and along with CELF4 they were also able to repress splicing of the NM (nonmuscle) exon both in vivo and in vitro. Activation of SM exon splicing was associated with displacement of PTB from the polypyrimidine tract by binding of CUG-BP at adjacent sites. Our data provides direct evidence for the activity of CELF proteins as both activators and repressors of splicing within a single-model system of alternative splicing, and suggests a model whereby alpha-actinin alternative splicing is regulated by synergistic and antagonistic interactions between members of the CELF and PTB families.     

ADAM15 is an adherens junction molecule whose surface expression can be driven by VE-cadherin

ADAM15 belongs to the family of proteins containing disintegrin and metalloprotease domains (ADAM) that have been implicated in cell adhesion via integrin binding and shedding of cell surface molecules. Here we provide the first report on the localization of an ADAM in adherens junctions. We show that ADAM15 colocalizes with a cell adhesion molecule, vascular endothelial (VE)-cadherin, which mediates endothelial cell adherens junction formation. In contrast, the distribution of ADAM15 correlates poorly with the localization in cell contacts of one of its proposed ligands, the beta1-integrin. Furthermore, ADAM15 accumulation in cell-cell contacts is preceded by VE-cadherin-mediated adherens junction formation. To investigate the dependence of ADAM15 surface expression on adherens junction formation, we coexpressed VE-cadherin with ADAM15 and an ADAM15 green fluorescence protein (GFP) fusion protein in Chinese hamster ovary cells. VE-cadherin coexpression results in the translocation of ADAM15-GFP to the cell periphery. Analysis of cell surface levels of ADAM15 and ADAM15-GFP, with or without VE-cadherin coexpression, clearly demonstrates that VE-cadherin can drive surface expression of ADAM15. Our data suggest that ADAM15 may be a novel component of adherens junctions and thus could play a role in endothelial functions that are mediated by these cell contacts.     

The translation of an antiapoptotic protein HIAP2 is regulated by an upstream open reading frame

HIAP2 is a multifunctional protein that is critically involved in the regulation of cell survival and apoptosis. Here, we show that HIAP2 5' untranslated region functions as a strong inhibitor of translation. Sequence analysis of human, mouse and rat sequences revealed that there exists a short open reading frame (ORF) that is located just upstream of the HIAP2 coding sequence. The translation of this uORF severely inhibited translation of the downstream reporter gene in vivo but not in vitro. Point mutation that destroys the CUG initiating codon of uORF markedly enhanced translation of the reporter gene without affecting the mRNA levels. Our results identify a novel translational regulatory mechanism that controls the expression of HIAP2 and point to the importance of tight regulation of antiapoptotic gene expression.     

Nuclear coded mitochondrial protein prohibitin is an iron regulated iron binding protein

Mitochondria are central to iron homeostasis. However, various proteins involved in iron metabolism inside the mitochondria are still to be identified. Herein we report that nuclear coded mitochondrial protein prohibitin binds to iron and involved in intracellular iron homeostasis. Like other iron regulated proteins, prohibitin mRNA contains functional iron-response element and is regulated by intracellular iron levels. Tyrosine residues involved in iron binding attribute of prohibitin are identified using site-directed mutagenesis. These data together suggest that prohibitin functions as an intracellular iron binding protein and plays a role in intracellular iron homeostasis.