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 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 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.

scholarly journals PTB Regulates the Processing of a 3′-Terminal Exon by Repressing both Splicing and Polyadenylation

2005 ◽  
Vol 25 (21) ◽  
pp. 9595-9607 ◽  
Author(s):  
Caroline Le Sommer ◽  
Michelle Lesimple ◽  
Agnès Mereau ◽  
Severine Menoret ◽  
Marie-Rose Allo ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Binding Protein ◽  
Specific Pattern ◽  
Polypyrimidine Tract ◽  
Terminal Exon ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Physiological Regulator

ABSTRACT The polypyrimidine tract binding protein (PTB) has been described as a global repressor of regulated exons. To investigate PTB functions in a physiological context, we used a combination of morpholino-mediated knockdown and transgenic overexpression strategies in Xenopus laevis embryos. We show that embryonic endoderm and skin deficient in PTB displayed a switch of the α-tropomyosin pre-mRNA 3′ end processing to the somite-specific pattern that results from the utilization of an upstream 3′-terminal exon designed exon 9A9′. Conversely, somitic targeted overexpression of PTB resulted in the repression of the somite-specific exon 9A9′ and a switch towards the nonmuscle pattern. These results validate PTB as a key physiological regulator of the 3′ end processing of the α-tropomyosin pre-mRNA. Moreover, using a minigene strategy in the Xenopus oocyte, we show that in addition to repressing the splicing of exon 9A9′, PTB regulates the cleavage/polyadenylation of this 3′-terminal exon.

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