Abstract 174: Regulation of Kv11.1 C-terminal Isoform Expression by Polypyrimidine Tract Binding Protein

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Qiuming Gong ◽  
Anastasiya Goldys ◽  
Zhengfeng Zhou
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Developmental Regulation ◽  
Delayed Rectifier ◽  
Polypyrimidine Tract ◽  
Rnase Protection ◽  
Polypyrimidine Tract Binding ◽  
Alternative Processing ◽  
Polypyrimidine Tract Binding Protein ◽  
Isoform Expression

The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative processing. Intron 9 splicing leads to the formation of a functional, full-length Kv11.1a isoform, and polyadenylation within intron 9 generates a non-functional, C-terminally truncated Kv11.1a-USO isoform. In this study we investigated the developmental regulation of Kv11.1 isoform expression. We showed that Kv11.1a expression was lower than that of Kv11.1a-USO in the adult heart, but the levels of Kv11.1a and Kv11.1a-USO were similar in the fetal heart. We studied the effect of polypyrimidine tract binding protein (PTB) on the alternative processing of KCNH2 pre-mRNA. PTB is an RNA-binding protein well known for its role in the regulation of alternative splicing. Recently, PTB has been shown to regulate polyadenylation. We showed that PTB increased Kv11.1a isoform expression and decreased Kv11.1a-USO isoform expression by the RNase protection assay and immunoblot analysis. In patch-clamp experiments, we found that PTB significantly increased Kv11.1 current. Our findings suggest that the relative expression of Kv11.1 C-terminal isoforms can be regulated by PTB. It has been reported that PTB protein abundance is progressively reduced during postnatal heart development. Thus, PTB may play an important role in developmental regulation of Kv11.1 isoform expression in the heart.

scholarly journals Nucleocytoplasmic Shuttling of Polypyrimidine Tract-binding Protein Is Uncoupled from RNA Export

2001 ◽  
Vol 12 (12) ◽  
pp. 3808-3820 ◽  
Author(s):  
Rajesh V. Kamath ◽  
Daniel J. Leary ◽  
Sui Huang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Binding Protein ◽  
Dependent Manner ◽  
Polypyrimidine Tract ◽  
Nucleocytoplasmic Shuttling ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Rna Export

Polypyrimidine tract binding protein, PTB/hnRNP I, is involved in pre-mRNA processing in the nucleus and RNA localization and translation in the cytoplasm. In this report, we demonstrate that PTB shuttles between the nucleus and cytoplasm in an energy-dependent manner. Deletion mutagenesis demonstrated that a minimum of the N terminus and RNA recognition motifs (RRMs) 1 and 2 are necessary for nucleocytoplasmic shuttling. Deletion of RRM3 and 4, domains that are primarily responsible for RNA binding, accelerated the nucleocytoplasmic shuttling of PTB. Inhibition of transcription directed by either RNA polymerase II alone or all RNA polymerases yielded similar results. In contrast, selective inhibition of RNA polymerase I did not influence the shuttling kinetics of PTB. Furthermore, the intranuclear mobility of GFP-PTB, as measured by fluorescence recovery after photobleaching analyses, increased significantly in transcriptionally inactive cells compared with transcriptionally active cells. These observations demonstrate that nuclear RNA transcription and export are not necessary for the shuttling of PTB. In addition, binding to nascent RNAs transcribed by RNA polymerase II and/or III retards both the nuclear export and nucleoplasmic movement of PTB. The uncoupling of PTB shuttling and RNA export suggests that the nucleocytoplasmic shuttling of PTB may also play a regulatory role for its functions in the nucleus and cytoplasm.

Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein

2008 ◽  
Vol 36 (4) ◽  
pp. 641-647 ◽  
Author(s):  
Kirsty Sawicka ◽  
Martin Bushell ◽  
Keith A. Spriggs ◽  
Anne E. Willis
Keyword(s):  
Translation Initiation ◽  
Cellular Localization ◽  
Rna Binding ◽  
Binding Protein ◽  
Control Cell ◽  
Rna Binding Protein ◽  
Alternative Form ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein

PTB (polypyrimidine-tract-binding protein) is a ubiquitous RNA-binding protein. It was originally identified as a protein with a role in splicing but it is now known to function in a large number of diverse cellular processes including polyadenylation, mRNA stability and translation initiation. Specificity of PTB function is achieved by a combination of changes in the cellular localization of this protein (its ability to shuttle from the nucleus to the cytoplasm is tightly controlled) and its interaction with additional proteins. These differences in location and trans-acting factor requirements account for the fact that PTB acts both as a suppressor of splicing and an activator of translation. In the latter case, the role of PTB in translation has been studied extensively and it appears that this protein is required for an alternative form of translation initiation that is mediated by a large RNA structural element termed an IRES (internal ribosome entry site) that allows the synthesis of picornaviral proteins and cellular proteins that function to control cell growth and cell death. In the present review, we discuss how PTB regulates these disparate processes.

scholarly journals Solution and crystal structures of a C-terminal fragment of the neuronal isoform of the polypyrimidine tract binding protein (nPTB)

2014 ◽  
Author(s):  
Amar Joshi ◽  
Vicent Esteve ◽  
Adrian N Buckroyd ◽  
Markus Blatter ◽  
Frédéric HT Allain ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Solution Structure ◽  
Rna Binding ◽  
Binding Protein ◽  
Amino Acid Side Chain ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein

The eukaryotic polypyrimidine tract binding protein (PTB) serves primarily as a regulator of alternative splicing of messenger RNA, but is also co-opted to other roles such as RNA localisation and translation initiation from internal ribosome entry sites. The neuronal paralogue of PTB (nPTB) protein is 75% identical in amino acid sequence with PTB. Although the two proteins have broadly similar RNA binding specificities and effects on RNA splicing, differential expression of PTB and nPTB can lead to the generation of alternatively spliced mRNAs. RNA binding by PTB and nPTB is mediated by four RNA recognition motifs (RRMs). We present here the crystal and solution structures of the C-terminal domain of nPTB (nPTB34) which contains RRMs 3 and 4. As expected the structures are similar to each other and to the solution structure of the equivalent fragment from PTB (PTB34). The result confirms that, as found for PTB, RRMs 3 and 4 of nPTB interact with one another to form a stable unit that presents the RNA-binding surfaces of the component RRMs on opposing sides. The major differences between PTB34 and nPTB34 arise from amino acid side chain substitutions on the exposed β-sheet surfaces and adjoining loops of each RRM, which are likely to modulate interactions with RNA.

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 Functional interactions between polypyrimidine tract binding protein and PRI peptide ligand containing proteins

2016 ◽  
Vol 44 (4) ◽  
pp. 1058-1065 ◽  
Author(s):  
Miguel B. Coelho ◽  
David B. Ascher ◽  
Clare Gooding ◽  
Emma Lang ◽  
Hannah Maude ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Dorsal Surface ◽  
Peptide Ligand ◽  
Rna Recognition Motif ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Binding Domains ◽  
Polypyrimidine Tract Binding Protein ◽  
Nuclear Ribonucleoprotein

Polypyrimidine tract binding protein (PTBP1) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that plays roles in most stages of the life-cycle of pre-mRNA and mRNAs in the nucleus and cytoplasm. PTBP1 has four RNA binding domains of the RNA recognition motif (RRM) family, each of which can bind to pyrimidine motifs. In addition, RRM2 can interact via its dorsal surface with proteins containing short peptide ligands known as PTB RRM2 interacting (PRI) motifs, originally found in the protein Raver1. Here we review our recent progress in understanding the interactions of PTB with RNA and with various proteins containing PRI ligands.

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 Solution and crystal structures of a C-terminal fragment of the neuronal isoform of the polypyrimidine tract binding protein (nPTB)

2014 ◽  
Author(s):  
Amar Joshi ◽  
Vicent Esteve ◽  
Adrian N Buckroyd ◽  
Markus Blatter ◽  
Frédéric HT Allain ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Solution Structure ◽  
Rna Binding ◽  
Binding Protein ◽  
Amino Acid Side Chain ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein

The eukaryotic polypyrimidine tract binding protein (PTB) serves primarily as a regulator of alternative splicing of messenger RNA, but is also co-opted to other roles such as RNA localisation and translation initiation from internal ribosome entry sites. The neuronal paralogue of PTB (nPTB) protein is 75% identical in amino acid sequence with PTB. Although the two proteins have broadly similar RNA binding specificities and effects on RNA splicing, differential expression of PTB and nPTB can lead to the generation of alternatively spliced mRNAs. RNA binding by PTB and nPTB is mediated by four RNA recognition motifs (RRMs). We present here the crystal and solution structures of the C-terminal domain of nPTB (nPTB34) which contains RRMs 3 and 4. As expected the structures are similar to each other and to the solution structure of the equivalent fragment from PTB (PTB34). The result confirms that, as found for PTB, RRMs 3 and 4 of nPTB interact with one another to form a stable unit that presents the RNA-binding surfaces of the component RRMs on opposing sides. The major differences between PTB34 and nPTB34 arise from amino acid side chain substitutions on the exposed β-sheet surfaces and adjoining loops of each RRM, which are likely to modulate interactions with RNA.

scholarly journals Determination of functional domains in polypyrimidine-tract-binding protein

1998 ◽  
Vol 331 (1) ◽  
pp. 169-175 ◽  
Author(s):  
Young L. OH ◽  
Bumsuk HAHM ◽  
Yoon K. KIM ◽  
Hae K. LEE ◽  
Joo W. LEE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Binding Protein ◽  
Biochemical Properties ◽  
Polypyrimidine Tract ◽  
Terminal Part ◽  
Entry Site ◽  
Cytoplasmic Factor ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein

Polypyrimidine-tract-binding protein (PTB) is involved in pre-mRNA splicing and internal-ribosomal-entry-site-dependent translation. The biochemical properties of various segments of PTB were analysed in order to understand the molecular basis of the PTB functions. The protein exists in oligomeric as well as monomeric form. The central part of PTB (amino acids 169–293) plays a major role in the oligomerization. PTB contains several RNA-binding motifs. Among them, the C-terminal part of PTB (amino acids 329–530) exhibited the strongest RNA-binding activity. The N-terminal part of PTB is responsible for the enhancement of RNA binding by HeLa cell cytoplasmic factor(s).

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