Frequent co-regulation of splicing and polyadenylation by RNA-binding proteins inferred with MAPP

Maturation of eukaryotic pre-mRNAs via splicing, 3' end cleavage and polyadenylation is modulated across cell types and conditions by a variety of RNA-binding proteins (RBPs). Although over 1'500 proteins are associated with RNAs in human cells, their binding motifs, targets and functions still remain to be elucidated, especially in the complex environment of human tissues and in the context of diseases. To overcome the lack of methods for systematic and automated detection of sequence motif-guided changes in pre-mRNA processing based on RNA sequencing (RNA-seq) data we have developed MAPP (Motif Activity on Pre-mRNA Processing). We demonstrate MAPP's functionality by applying it to RNA-seq data from 284 RBP knock-down experiments in the ENCODE project, from which MAPP not only infers position-dependent impact profiles of known regulators, but also reveals RBPs that modulate both the inclusion of cassette exons and the poly(A) site choice. Among these, the Polypyrimidine Tract Binding Protein 1 (PTBP1) has a similar activity in glioblastoma samples. This highlights the ability of MAPP to unveil global regulators of mRNA processing under physiological and pathological conditions.

LncRNA GAS5 Inhibits Temozolomide Chemoresistance to Glioma Via Inactivating Wnt/β-Catenin Pathway by Interacting with PTBP1

Purpose Temozolomide-based therapeutic resistance has become the crucial cause of chemotherapy failure in glioma treatment. Long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) is reported to be downregulated in glioma and to inhibit tumor progression and metastasis. This study aimed to investigate function and potential regulatory mechanism of GAS5 in temozolomide (TMZ) chemoresistance to glioma.Methods qRT-PCR, western blotting and immunofluorescence were used to measure the levels of GAS5 and proteins. RNA-binding protein immunoprecipitation assay was used to analyze the interaction between GAS5 and PTBP1. Flow cytometry apoptosis assay, CCK-8 assay, colony formation assay and nude mice xenograft experiments were used to detect the effects of GAS5 on TMZ resistance in glioma.Results Downregulation of GAS5 might predict a poor prognosis in glioma patients. Overexpression of GAS5 improves the sensitivity to TMZ in glioma cells. Mechanistically, GAS5 could interact with polypyrimidine tract binding protein 1 (PTBP1) to downregulate its expression, thereby inactivating the Wnt/β-catenin pathway. Moreover, GAS5 could increase the anti-tumor effect of TMZ in vivo. Conclusion This study indicated that GAS5 contributed to TMZ chemoresistance of glioma through interacting with PTBP1, and then inhibiting Wnt/β-catenin pathway, which provides a novel approach to develop promising therapeutic strategy.

Repressing PTB is incapable to convert reactive astrocytes to dopaminergic neurons in a mouse model of Parkinson's disease

Lineage reprograming of resident glia cells to induced dopaminergic neurons (iDAns) holds attractive prospect for cell-replacement therapy of Parkinson's disease (PD). Recently, whether repressing polypyrimidine tract binding protein (PTB) could truly achieve efficient astrocyte-to-iDAn conversion in substantia nigra and striatum aroused widespread controversy. Although reporter+ iDAns were observed by two groups after delivering adeno-associated virus (AAV) expressing a reporter with shRNA or Crispr-CasRx to repress astroglial PTB, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage tracing method. By adopting stringent lineage tracing strategy, two other studies showed that neither knockdown nor genetic deletion of quiescent astroglial PTB fails to obtain iDAns under physiological condition. However, the role of reactive astrocyte might be underestimated since upon brain injury, reactive astrocyte could acquire certain stem cell hallmarks which may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTB repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2-mediated specific astrocyte-lineage tracing method to investigate whether reactive astrocytes could be converted to DAns in the 6-OHDA PD model. However, we found that no astrocyte-originated DAns was generated after effective knockdown of astroglial PTB either in the substantia nigra or in the striatum, while AAV leakage to nearby neurons was observed. Our results further confirmed that repressing PTB is unable to convert astrocytes to DA neurons no matter in physiological or PD-related pathological conditions.

Ptbp1 deletion does not induce glia-to-neuron conversion in adult mouse retina and brain.

Somatic reprogramming of glia into neurons is a potentially promising approach for the replacement of neurons lost to injury or neurodegenerative disorders. Knockdown of the polypyrimidine tract-binding protein Ptbp1 has been recently reported to induce efficient conversion of retinal Mϋller glia and brain astrocytes into functional neurons. However, genetic analysis of Ptbp1 function in adult glia has not been conducted. Here, we use a combination of genetic lineage tracing, scRNA-Seq, and electrophysiological analysis to show that specific deletion of Ptbp1 in adult retinal Mϋller glia and brain astrocytes does not lead to any detectable level of glia-to-neuron conversion. Few changes in gene expression are observed in glia following Ptbp1 deletion, and glial identity is maintained. These findings highlight the importance of using genetic manipulation and lineage tracing methods in studying cell type conversion.

THE SPLICING FACTOR PTBP1 REPRESSES TP63 γ ISOFORM PRODUCTION IN SQUAMOUS CELL CARCINOMA

The TP63 gene encodes the transcription factor p63. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, β, γ and δ. The regulatory functions of p63 may be isoform-specific. The α isoform inhibits the epithelial to mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Here, we observed in TCGA data that a high ratio of the TP63γ isoform to the other isoforms is a pejorative factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC). We therefore addressed the regulation of the γ isoform. In several tissues (GTEX data), the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines leads to an increase in abundance of the γ isoform. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63γ-specific exon. The region around the TP63γ-specific exon was sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Finally, we demonstrated that the regulation of TP63γ production by PTBP1 is conserved in amphibians, revealing that it encounters a strong evolutionary pressure. Together, these results identify TP63γ as a prognostic marker in HNSCC, and identify PTBP1 as a direct negative regulator of its production.

Evaluation of Angelica decursiva reference genes under various stimuli for RT-qPCR data normalization

Angelica decursiva is one of the lending traditional Chinese medicinal plants producing coumarins. Notably, several studies have focused on the biosynthesis and not the RT-qPCR (quantitative real-time reverse transcription polymerase chain reaction) study of coumarins. This RT-qPCR technique has been extensively used to investigate gene expression levels in plants and the selection of reference genes which plays a crucial role in standardizing the data form the RT-qPCR analysis. In our study, 11 candidate reference genes were selected from the existing transcriptome data of Angelica decursiva. Here, four different types of statistical algorithms (geNorm, NormFinder, BestKeeper, and Delta Ct) were used to calculate and evaluate the stability of gene expression under different external treatments. Subsequently, RefFinder analysis was used to determine the geometric average of each candidate gene ranking, and to perform comprehensive index ranking. The obtained results showed that among all the 11 candidate reference genes, SAND family protein (SAND), protein phosphatase 2A gene (PP2A), and polypyrimidine tract-binding protein (PTBP) were the most stable reference genes, where Nuclear cap binding protein 2 (NCBP2), TIP41-like protein (TIP41), and Beta-6-tubulin (TUBA) were the least stable genes. To the best of our knowledge, this work is the first to evaluate the stability of reference genes in the Angelica decursiva which has provided an important foundation on the use of RT-qPCR for an accurate and far-reaching gene expression analysis in this medicinal plant.

PTBP3 Modulates of P53 Expression and Promotes Colorectal Cancer Cell Proliferation by Maintaining UBE4A mRNA Stability

Background Colorectal cancer (CRC) is the most common malignancy worldwide and has become the second leading cause of cancer-related death worldwide. The RNA-binding protein polypyrimidine tract-binding protein 3 (PTBP3) was recently reported to play a critical role in multiple cancers, and its molecular mechanisms involve RNA splicing, 3′ end processing and translation. However, the role of PTBP3 in CRC is unclear. Methods We analyzed the expression levels of PTPB3 using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets and clinical tissues. The effect of PTBP3 on CRC cell proliferation was measured by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry and tumor xenograft assays. A series of experiments were conducted to reveal the mechanisms by which PTBP3 promotes CRC proliferation. Results We showed that PTBP3 was upregulated in CRC and associated with a poor prognosis. Knockdown of PTBP3 in CRC cell lines restricted CRC proliferative capacities in vitro and in vivo. Mechanistically, we found that PTBP3 regulated the expression of the E3 ubiquitin ligase ubiquitination factor E4A (UBE4A) by binding the 3' untranslated region (UTR) of its mRNA, thereby preventing its degradation. We also discovered that UBE4A participated in the degradation of P53, and knockdown of PTBP3 in CRC cell lines increased P53 expression. Overexpression of UBE4A rescued PTBP3 knockdown-induced inhibition of CRC cell proliferation and P53 expression. Conclusions PTBP3 plays an essential role in CRC cell proliferation by stabilizing UBE4A to regulate P53 expression and may serve as a new prognostic biomarker and effective therapeutic target for CRC.

Role of Long Non-Coding RNAs and the Molecular Mechanisms Involved in Insulin Resistance

Long non-coding RNAs (lncRNAs) are single-stranded RNA biomolecules with a length of >200 nt, and they are currently considered to be master regulators of many pathological processes. Recent publications have shown that lncRNAs play important roles in the pathogenesis and progression of insulin resistance (IR) and glucose homeostasis by regulating inflammatory and lipogenic processes. lncRNAs regulate gene expression by binding to other non-coding RNAs, mRNAs, proteins, and DNA. In recent years, several mechanisms have been reported to explain the key roles of lncRNAs in the development of IR, including metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), imprinted maternal-ly expressed transcript (H19), maternally expressed gene 3 (MEG3), myocardial infarction-associated transcript (MIAT), and steroid receptor RNA activator (SRA), HOX transcript antisense RNA (HOTAIR), and downregulated Expression-Related Hexose/Glucose Transport Enhancer (DREH). LncRNAs participate in the regulation of lipid and carbohydrate metabolism, the inflammatory process, and oxidative stress through different pathways, such as cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), polypyrimidine tract-binding protein 1/element-binding transcription factor 1c (PTBP1/SREBP-1c), AKT/nitric oxide synthase (eNOS), AKT/forkhead box O1 (FoxO1), and tumor necrosis factor-alpha (TNF-α)/c-Jun-N-terminal kinases (JNK). On the other hand, the mechanisms linked to the molecular, cellular, and biochemical actions of lncRNAs vary according to the tissue, biological species, and the severity of IR. Therefore, it is essential to elucidate the role of lncRNAs in the insulin signaling pathway and glucose and lipid metabolism. This review analyzes the function and molecular mechanisms of lncRNAs involved in the development of IR.

Tobacco Smoking Increases Methylation of Polypyrimidine Tract Binding Protein 1 Promoter in Intracranial Aneurysms

DNA methylation at the gene promoter region is reportedly involved in the development of intracranial aneurysm (IA). This study aims to investigate the methylation levels of polypyrimidine tract-binding protein 1 (PTBP1) in IA, as well as its potential to predict IA. Forty-eight patients with IA and 48 age- and sex-matched healthy controls were recruited into this study. Methylation levels of CpG sites were determined via bisulfite pyrosequencing. The PTBP1 levels in the blood were determined using a real-time quantitative reverse transcription-polymerase chain reaction test. Significant differences were found between IAs and controls in CpG1 (p = 0.001), CpG2 (p < 0.001), CpG3 (p = 0.037), CpG4 (p = 0.003), CpG5 (p = 0.006), CpG6 (p = 0.02), and mean methylation (p < 0.001). The mRNA level of PTBP1 in the blood was much lower in IAs compared with controls (p = 0.002), and the PTBP1 expression was significantly associated with DNA methylation promoter levels in individuals (r = −0.73, p < 0.0001). In addition, stratification analysis comparing smokers and non-smokers revealed that tobacco smokers had significantly higher levels of DNA methylation in PTBP1 than non-smokers (p = 0.002). However, no statistical difference in PTBP1 methylation was found between ruptured and unruptured IA groups (p > 0.05). The ROC analyses of curves revealed that PTBP1 methylation may be a predictor of IA regardless of sex (both sexes, area under curve (AUC) = 0.78, p < 0.0001; male, AUC = 0.76, p = 0.002; female, AUC = 0.79, p < 0.0001). These findings suggest that long-term tobacco smoke exposure led to DNA methylation in the promoter region of the PTBP1 gene, which further decreased PTBP1 gene expression and participated in the pathogenesis of IA. The methylation of PTBP1 may be a potential predictive marker for the occurrence of IA.