Alternative splicing analysis showed the splicing factor polypyrimidine tract-binding protein 1 as a potential target in acute myeloid leukemia therapy.

Alternative splicing (AS) is a universal post-transcriptional regulation process in cells, and increasing evidences have validated its crucial role in tumors. We collected AS event, gene expression, and clinical data of 178 AML patients from The Cancer Genome Atlas (TCGA) project. More than 1,000 AS events were found associated with overall survival (OS), and alternate promoter (AP) events were the most significant. The expression of the KIAA0930 transcript was the most significantly different AS event selected from AP events and significantly correlated with the expression of the splicing factor (SF) polypyrimidine tract-binding protein 1 (PTBP1). Then, the roles of PTBP1 on AS of the KIAA0930 and the proliferation of AML cells were confirmed. KIAA0930 variant 1 (KIAA0930-1) was upregulated and variant 2 (KIAA0930-2) downregulated with knockdown PTBP1 expression of AML cells by specific shRNA. A low level of PTBP1 can decrease the proliferation ability of AML cells. In conclusion, the results showed that PTBP1 might be a potential target for AML therapy.

LncRNA-Malat1 is Involved in Lipotoxicity-Induced ß-cell Dysfunction and the Therapeutic Effect of Exendin-4 via Ptbp1.

Increasing evidence indicates that long noncoding RNAs (lncRNAs) have crucial roles in various biological processes. However, the contribution of lncRNAs to ß-cell dysfunction and their roles in diabetes therapeutics remain poorly understood. The aim of this study was to identify the lncRNAs dysregulated in diabetic islets and to explore the lncRNAs involved in ß-cell function as potential therapeutic targets. By using RNA sequencing and real-time PCR, we identified thousands of lncRNAs in the islets of db/db mice and db/m littermate mice. Among the differentially expressed lncRNAs, lncRNA-Malat1 (metastasis-associated lung adenocarcinoma transcript 1) was reduced in the islets of db/db mice and palmitate-treated MIN6 cells. The results of TUNEL, Western blot and flow cytometric analyses, and GSIS assays revealed that Malat1 knockdown significantly induced ß-cell apoptosis and inhibited insulin secretion. Mechanistically, RNA immunoprecipitation showed that Malat1 enhanced polypyrimidine tract-binding protein 1 (Ptbp1) protein stability by direct interaction, thereby adjusting the ratio of pyruvate kinase muscle (PKM) isoforms 1 and 2 (PKM1/PKM2). Moreover, luciferase assay and chromatin immunoprecipitation indicated that Malat1 was transcriptionally activated by pancreatic and duodenal homeobox 1 (Pdx1), through which exendin-4 alleviated lipotoxicity-induced ß-cell damage. In summary, our findings suggested the involvement of Malat1 in ß-cell dysfunction under diabetic conditions via the Malat1/Ptbp1/PKM2 pathway. In addition, exendin-4 ameliorated ß-cell impairment by Pdx1-mediated Malat1 upregulation. Hence, Malat1 may serve as a therapeutic target for the treatment of type 2 diabetes.

Inhibition of polypyrimidine tract-binding protein 3 induces apoptosis and cell cycle arrest, and enhances the cytotoxicity of 5- fluorouracil in gastric cancer cells.

BACKGROUND: Human polypyrimidine tract binding protein 3 (PTBP3) was first discovered in 1999 and has been well characterised as a differentiation regulator. However, its role in human cancer has rarely been reported. Our previous study revealed increased PTBP3 protein level in gastric cancer tissues. Downregulation of PTBP3 suppressed the proliferation and differentiation of gastric cancer cells in vivo. METHODS: PTBP3 mRNA levels in human gastric cancer and adjuvant non-tumour tissues were detected. Apoptosis and 5-FU effect were determined in PTBP3-silenced gastric cancer cells. Underlying molecular mechanisms were investigated. RESULTS: MRNA expression of PTBP3 was upregulated in gastric cancer tissues, especially in those at an advanced stage. PTBP3 silencing led to apoptosis, under which modulation of PTB and thereby switch of Bcl-x pre-mRNA splicing pattern might be an important mechanism. Further research found that inhibition of PTBP3 expression enhanced the chemosensitivity of gastric cancer cells towards 5-FU treatment. This was mediated by reduced expression of histone deacetylase 6 (HDAC6), which further inhibited the phosphorylation of Akt and the expression of thymidylate synthase (TYMS), the critical determinant of 5-FU cytotoxicity. CONCLUSIONS: PTBP3 might serve as a biomarker of gastric cancer or potential target for anti-cancer therapy.

Polypyrimidine tract-binding proteins of potato mediate tuberization through an interaction with StBEL5 RNA.

Polypyrimidine tract-binding (PTB) proteins are a family of RNA-binding proteins that function in a wide range of RNA metabolic processes by binding to motifs rich in uracils and cytosines. A PTB protein of pumpkin was identified as the core protein of an RNA-protein complex that trafficks RNA. The biological function of the PTB-RNA complex, however, has not been demonstrated. In potato, six PTB proteins have been identified, and two, designated StPTB1 and StPTB6, are similar to the phloem-mobile pumpkin type. RNA binding assays confirmed the interaction of StPTB1 and StPTB6 with discrete pyrimidine-rich sequences of the 3'-untranslated regions of the phloem-mobile mRNA, StBEL5. The promoter of StPTB1 was active in companion cells of phloem in both stem and petioles. Expression of both types was evident in phloem cells of roots and in stolons during tuber formation. RNA accumulation of both PTB proteins was induced by short days in leaves in correlation with enhanced accumulation of StBEL5 RNA. StPTB suppression lines exhibited reduced tuber yields and decreased StBEL5 RNA accumulation, whereas StPTB overexpression lines displayed an increase in tuber production correlated with the enhanced production in stolons of steady-state levels of StBEL5 transcripts and RNA of key tuber identity genes. In StPTB overexpression lines, both the stability and long-distance transport of StBEL5 transcripts were enhanced, whereas in suppression lines stability and transport decreased. Using a transgenic approach, it is shown that the StPTB family of RNA-binding proteins regulate specific stages of development through an interaction with phloem-mobile transcripts of StBEL5.

PTBP-dependent PSD-95 and CamKIIα alternative splicing in the lens.

PURPOSE: Parallels described between neurons and lens fiber cells include detailed similarities in sub-cellular structures that increasingly show shared expression of genes involved in the construction and function of these structures in neurons. Intriguingly, associated modes of molecular regulation of these genes that had been thought to distinguish neurons have been identified in the lens as well. Both elongated cell types form membrane protrusions with similar size, shape, and spacing that exclude microtubules, contain F-actin, and are coated with the clathrin/AP-2 adaptor. Lenses express glutamate and gamma-aminobutyric acid (GABA) receptors with signaling and channel proteins shown to act together at neuronal membranes. Postsynaptic density protein 95 (PSD-95) and Ca(2+)/calmodulin-dependent protein kinase (CaMKIIα) expression and functions illustrate the integration of aspects of neuronal molecular and cell biology and were investigated here in the lens. METHODS: Immunofluorescence, immunoblot, and RT-PCR methods were used to assess protein expression and alternative transcript splicing. RESULTS: We showed the essential dendritic spine scaffold protein PSD-95 is expressed in lenses and demonstrated lens PSD-95 transcripts undergo polypyrimidine tract binding protein (PTBP)-dependent alternative splicing of its pivotal exon 18 required to avoid nonsense-mediated decay, and showed PTBP-dependent alternative splicing of CaMKIIα transcripts in the lens. The PSD-95 protein was observed at fiber cell membranes overlapping with N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate and GABA receptor proteins, tyrosine phosphatase STEP, CaMKIIα, the Ca(V)1.3 calcium channel, and clathrin, which were previously identified at lens fiber cell membranes. During neurogenesis, miR-124 is expressed that suppresses PTBP1 and promotes these splicing events. miR-124 is also expressed in mammalian lenses and upregulated during lens regeneration in amphibians, consistent with previous demonstrations of PTBP1,2 and PTBP-dependent PTBP2 exon 10 splicing in rodent lenses. CONCLUSIONS: Findings of this dendritic spine scaffold protein and conservation of its key mode of molecular regulation in the lens provides further evidence that key aspects of the neuron morphogenetic program are shared with the lens.

Mapping and characterization of the interaction interface between two polypyrimidine-tract binding proteins and a nova-type protein of Solanum tuberosum.

Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that generally contain four RNA recognition motifs (RRMs). In potato, six cDNAs encoding full-length PTB proteins have been identified. In the present study Nova1-like protein, designated StNova1, was identified as a potential interacting partner of the StPTB proteins via yeast two-hybrid screening. Nova protein is a RNA-binding protein that contains three K-homology (KH) domains. In humans, these proteins are involved in regulation of neuronal RNA metabolism but the role of Nova-like proteins in plants is poorly understood. We have validated this interaction and mapped the protein binding region on StNova1 and StPTB1 and -6 using a novel domain interaction phage display (DIPP) technique. The interaction between the two RNA-binding proteins StPTB1/6 and StNova1 is mediated through linker regions that are distinctly separated from the RRMs. Furthermore, using a random 21-mer phage-peptide library, we have identified a number of peptides with the consensus sequence motif [S/G][V/I][L/V]G that recognize the StPTB proteins. One over-represented peptide that recognizes StPTB6 contains the GVLGPWP sequence that is similar to the GIGGRYP sequence in the glycine-rich linker region between the KH2 and KH3 domains of StNova1. We show, through site-specific mutations, the importance of glycine and proline residues in StNova1-StPTB interactions.

Promoter activity of polypyrimidine tract-binding protein genes of potato responds to environmental cues.

Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that target specific RNAs for post-transcriptional processing by binding cytosine/uracil motifs. PTBs have established functions in a range of RNA processes including splicing, translation, stability and long-distance transport. Six PTB-like genes identified in potato have been grouped into two clades based on homology to other known plant PTBs. StPTB1 and StPTB6 are closely related to a PTB protein discovered in pumpkin, designated CmRBP50, and contain four canonical RNA-recognition motifs. CmRBP50 is expressed in phloem tissues and functions as the core protein of a phloem-mobile RNA/protein complex. Sequence from the potato genome database was used to clone the upstream sequence of these two PTB genes and analyzed to identify conserved cis-elements. The promoter of StPTB6 was enriched for regulatory elements for light and sucrose induction and defense. Upstream sequence of both PTB genes was fused to ß-glucuronidase and monitored in transgenic potato lines. In whole plants, the StPTB1 promoter was most active in leaf veins and petioles, whereas StPTB6 was most active in leaf mesophyll. Both genes are active in new tubers and tuber sprouts. StPTB6 expression was induced in stems and stolon sections in response to sucrose and in leaves or petioles in response to light, heat, drought and mechanical wounding. These results show that CmRBP50-like genes of potato exhibit distinct expression patterns and respond to both developmental and environmental cues.

Involvement of polypyrimidine tract-binding protein (PTB)-related proteins in pollen germination in Arabidopsis.

The pollen grains of most angiosperms contain stores of RNAs and their translation products required for pollen germination and subsequent early elongation of pollen tubes. Polypyrimidine tract-binding protein (PTB), which is involved in the regulation of pre-mRNA alternative splicing, internal ribosomal entry site (IRES)-mediated translation and mRNA localization/sorting, is known to act as a bridging molecule between RNAs and a variety of cellular factors to fulfill cellular functions in both the nucleus and cytoplasm. Moreover, it has been reported that PTB plays roles in the differentiation and development of animal cells and tissues. In the Arabidopsis genome, there are two PTB-related genes, tentatively termed AtPTB1 and AtPTB2. In the present study, the physiological functions of AtPTBs were investigated using genetic and cytological approaches. The AtPTB promoter was highly active in vegetative cells of mature pollen grains, and AtPTB was localized in the nucleus and cytoplasm of these vegetative cells. Mutations in the AtPTB genes resulted in decreased germination efficiency, and this effect was rescued by introduction of the AtPTB2 promoter::AtPTB2-GFP. Taken together, these findings suggest that AtPTB is involved in pollen germination through possible RNA metabolism processes in late-maturing and mature pollen grains.

Localization of polypyrimidine-tract-binding protein is involved in the regulation of albumin synthesis by branched-chain amino acids in HepG2 cells.

Long-term supplementation of branched-chain amino acids (BCAA) improves hypoalbuminemia in patients with cirrhosis. Our previous findings have suggested that the binding of polypyrimidine-tract-binding protein (PTB) to rat albumin mRNA attenuates its translation. The aim of the present study was to investigate the role of PTB in the regulation of albumin synthesis by BCAA in human hepatoma cells. HepG2 cells were cultured in a medium containing no amino acids (AA-free medium), a medium containing only 1 amino acid (a BCAA: valine, leucine or isoleucine) or a medium containing all 20 amino acids (AA-complete medium). HepG2 cells cultured in AA-complete medium secreted much more albumin than cells cultured in AA-free medium, with no difference in albumin mRNA levels. In cells cultured in AA-free medium, nuclear export of PTB was observed, and the level of the albumin mRNA-PTB complex was greater than in cells cultured in AA-complete medium. Addition of amino acids stimulated nuclear import of PTB. However, addition of amino acids with rapamycin inhibited the nuclear import of PTB. The addition of leucine, but not of valine or isoleucine, to AA-free medium increased albumin secretion and stimulated the nuclear import of PTB. These data indicate that the mammalian target of rapamycin is involved in the regulation of PTB localization and that leucine promotes albumin synthesis by inhibiting the formation of the albumin mRNA-PTB complex.