scholarly journals Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo

2000 ◽  
Vol 20 (5) ◽  
pp. 1583-1595 ◽  
Author(s):  
Rainer Gosert ◽  
Ki Ha Chang ◽  
Rene Rijnbrand ◽  
MinKyung Yi ◽  
David V. Sangar ◽  
...  
Keyword(s):  
Binding Protein ◽  
Fold Increase ◽  
Null Mutant ◽  
Polypyrimidine Tract ◽  
Reporter Protein ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Independent Translation

ABSTRACT The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

scholarly journals Polypyrimidine Tract-Binding Protein Positively Regulates Inclusion of an Alternative 3′-Terminal Exon

1999 ◽  
Vol 19 (1) ◽  
pp. 78-85 ◽  
Author(s):  
Hua Lou ◽  
David M. Helfman ◽  
Robert F. Gagel ◽  
Susan M. Berget
Keyword(s):  
Binding Protein ◽  
Consensus Sequence ◽  
Polypyrimidine Tract ◽  
Terminal Exon ◽  
Exon Inclusion ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Exon 4 ◽  
A Site

ABSTRACT Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that 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. Here we show that 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 was 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 eliminated 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.

scholarly journals Protein Factor Requirements of the Apaf-1 Internal Ribosome Entry Segment: Roles of Polypyrimidine Tract Binding Protein and upstream of N-ras

2001 ◽  
Vol 21 (10) ◽  
pp. 3364-3374 ◽  
Author(s):  
Sally A. Mitchell ◽  
Emma C. Brown ◽  
Mark J. Coldwell ◽  
Richard J. Jackson ◽  
Anne E. Willis
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Cell Types ◽  
Polypyrimidine Tract ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Different Cell Types

ABSTRACT It has been reported previously that the 5′ untranslated region of the mRNA encoding Apaf-1 (apoptotic protease-activating factor 1) has an internal ribosome entry site (IRES), whose activity varies widely among different cell types. Here it is shown that the Apaf-1 IRES is active in rabbit reticulocyte lysates, provided that the system is supplemented with polypyrimidine tract binding protein (PTB) and upstream of N-ras (unr), two cellular RNA binding proteins previously identified to be required for rhinovirus IRES activity. In UV cross-linking assays and electrophoretic mobility shift assays with individual recombinant proteins, the Apaf-1 IRES binds unr but not PTB; however, PTB binding occurs if unr is present. Over a range of different cell types there is a broad correlation between the activity of the Apaf-1 IRES and their content of PTB and unr. In cell lines deficient in these proteins, overexpression of PTB and unr stimulated Apaf-1 IRES function. This is the first example where an IRES in a cellular mRNA has been shown to be functionally dependent, both in vitro and in vivo, on specific cellular RNA binding proteins. Given the critical role of Apaf-1 in apoptosis, these results have important implications for the control of the apoptotic cascade.

scholarly journals Differentiation-induced Colocalization of the KH-type Splicing Regulatory Protein with Polypyrimidine Tract Binding Protein and the c-srcPre-mRNA

2004 ◽  
Vol 15 (2) ◽  
pp. 774-786 ◽  
Author(s):  
Megan P. Hall ◽  
Sui Huang ◽  
Douglas L. Black
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Neuroblastoma Cell ◽  
Regulatory Protein ◽  
Neuroblastoma Cells ◽  
Neuroblastoma Cell Line ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein

We have examined the subcellular localization of the KH-type splicing regulatory protein (KSRP). KSRP is a multidomain RNA-binding protein implicated in a variety of cellular processes, including splicing in the nucleus and mRNA localization in the cytoplasm. We find that KSRP is primarily nuclear with a localization pattern that most closely resembles that of polypyrimidine tract binding protein (PTB). Colocalization experiments of KSRP with PTB in a mouse neuroblastoma cell line determined that both proteins are present in the perinucleolar compartment (PNC), as well as in other nuclear enrichments. In contrast, HeLa cells do not show prominent KSRP staining in the PNC, even though PTB labeling identified the PNC in these cells. Because both PTB and KSRP interact with the c-src transcript to affect N1 exon splicing, we examined the localization of the c-src pre-mRNA by fluorescence in situ hybridization. The src transcript is present in specific foci within the nucleus that are presumably sites of src transcription but are not generally perinucleolar. In normally cultured neuroblastoma cells, these src RNA foci contain PTB, but little KSRP. However, upon induced neuronal differentiation of these cells, KSRP occurs in the same foci with src RNA. PTB localization remains unaffected. This differentiation-induced localization of KSRP with src RNA correlates with an increase in src exon N1 inclusion. These results indicate that PTB and KSRP do indeed interact with the c-src transcript in vivo, and that these associations change with the differentiated state of the cell.

Investigation of interactions of polypyrimidine tract-binding protein with artificial internal ribosome entry segments

2005 ◽  
Vol 33 (6) ◽  
pp. 1483-1486 ◽  
Author(s):  
K.A. Spriggs ◽  
S.A. Mitchell ◽  
A.E. Willis
Keyword(s):  
Translation Initiation ◽  
Binding Protein ◽  
Regulation Of Gene Expression ◽  
Sequence Motif ◽  
Polypyrimidine Tract ◽  
Protein Motif ◽  
Internal Ribosome Entry ◽  
Eukaryotic Translation ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein

Most eukaryotic translation initiation is thought to be dependent on the 5′-cap structure of the mRNA. It is becoming apparent, however, that the mRNAs of many genes contain IRESs (internal ribosome entry segments) within the 5′-UTR (5′-untranslated region) that allow ribosomes to initiate translation independently of the 5′-cap. IRESs can enable the expression of these genes under conditions (such as viral infection, cellular stress and apoptosis) when cap-dependent translation initiation is compromised, and also provide a target for regulation of gene expression. Recent results from our laboratory and others suggest that 10% of mRNAs (∼4000 genes) use this mechanism to initiate translation. One of the central goals of those working in the field of translation is to identify the sequence motif(s) and proteins that are required for internal ribosome entry. We have identified recently a unique PTB (polypyrimidine tract-binding protein) motif (CCU)n that is present in a large subset of cellular IRESs, and the results suggest that PTB itself is involved either directly or indirectly in ribosome recruitment. Here, we describe further investigations of PTB with artificial sequences that harbour this motif.

scholarly journals Bag-1 Internal Ribosome Entry Segment Activity Is Promoted by Structural Changes Mediated by Poly(rC) Binding Protein 1 and Recruitment of Polypyrimidine Tract Binding Protein 1

2004 ◽  
Vol 24 (12) ◽  
pp. 5595-5605 ◽  
Author(s):  
Becky M. Pickering ◽  
Sally A. Mitchell ◽  
Keith A. Spriggs ◽  
Mark Stoneley ◽  
Anne E. Willis
Keyword(s):  
Binding Sites ◽  
Structural Model ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Polypyrimidine Tract ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Internal Ribosome Entry Segment ◽  
Entry Window

ABSTRACT We have shown previously that an internal ribosome entry segment (IRES) directs the synthesis of the p36 isoform of Bag-1 and that polypyrimidine tract binding protein 1 (PTB-1) and poly(rC) binding protein 1 (PCBP1) stimulate IRES-mediated translation initiation in vitro and in vivo. Here, a secondary structural model of the Bag-1 IRES has been derived by using chemical and enzymatic probing data as constraints on the RNA folding algorithm Mfold. The ribosome entry window has been identified within this structural model and is located in a region in which many residues are involved in base-pairing interactions. The interactions of PTB-1 and PCBP1 with their cognate binding sites on the IRES disrupt many of the RNA-RNA interactions, and this creates a largely unstructured region of approximately 40 nucleotides that could permit ribosome binding. Mutational analysis of the PTB-1 and PCBP1 binding sites suggests that PCBP1 acts as an RNA chaperone to open the RNA in the vicinity of the ribosome entry window while PTB-1 is probably an essential part of the preinitiation complex.

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