Project description:To assess the global impact of TGF-beta on alternative splicing, we conducted RNA-seq of total RNAs isolated from various lines of HeLa cells that were generated for this study and treated without or with TGF-beta and EGF. Using rMAT tool from the RNA-Seq data and annotation of transcripts in GTF format, we identified differential alternative splicing events between untreated and treated cell lines. Our study shows that the TGF-beta-mediated alternative splicing affects the protein products of a large number of genes enriched in pathways critical for EMT, cykeskeleton organization, and adherens junction signaling. PCBP1 was required for the most, if not all, alternative splicing events detected. SRA accession number: SRP061634, BioProject ID PRJNA290799. RNAs from Hela cells were treated with TGF-β and EGF, in triplicates and sequenced on HiSeq2000 with 2 samples pooled into one lane with Illumina TruSeq v3 chemistry.
Project description:To assess the global impact of TGF-beta on alternative splicing, we conducted RNA-seq of total RNAs isolated from various lines of HeLa cells that were generated for this study and treated without or with TGF-beta and EGF. Using rMAT tool from the RNA-Seq data and annotation of transcripts in GTF format, we identified differential alternative splicing events between untreated and treated cell lines. Our study shows that the TGF-beta-mediated alternative splicing affects the protein products of a large number of genes enriched in pathways critical for EMT, cykeskeleton organization, and adherens junction signaling. PCBP1 was required for the most, if not all, alternative splicing events detected.
Project description:We performed EGF treatment and hnRNP A1 knockdown in HeLa cells and analyzed alternative splicing patterns by high-throughput RNA sequencing.
Project description:To investigate whether U1C plays a role in splicing regulation in human system, we performed siRNA-mediated knockdown of U1C in HeLa cells and analyzed alternative splicing patterns by high-throughput RNA sequencing (RNAseq) RNAseq performed with poly(A)+ selected total RNA from U1C-knockdown and control-treated HeLa cells
Project description:To investigate whether U1C plays a role in splicing regulation in human system, we performed siRNA-mediated knockdown of U1C in HeLa cells and analyzed alternative splicing patterns by high-throughput RNA sequencing (RNAseq)
Project description:To study the potential targets and functions of STAU1 which might be related to neuropathic pain, we obtained STAU1-regulated transcriptome in HeLa cells. High-throughput RNA sequencing(RNA-seq) were performed for HeLa cells and control cells to define the comprehensive gene expression profiles and identify the genome-wide alternative splicing events regulated by STAU1.
Project description:Analysis of transcriptome post hypoxia and TGF-β treatment in breast cancer In order to explore the role of TGF-β signaling in mediating the alternative splicing program in breast cancer, we profiled the pre-mRNA splicing and mRNA gene expression upon hypoxia and TGF-β treatment using Human Transcriptome Array 2.0.
Project description:Pre-mRNA splicing is regulated by developmental and environmental cues, but little is known about how specific signals are transduced in mammalian cells to regulate this critical gene expression step. Here, we report massive reprogramming of alternative splicing in response to EGF signaling. By blocking individual branches in EGF signaling, we found that Akt activation plays a major role, while other branches, such as the JAK/STAT and ERK pathways, make minor contributions to EGF-induced splicing. Activated Akt next branches to the SRPK family of kinases specific for SR proteins, rather than mTOR, by inducing SRPK autophosphorylation that switches the splicing kinases from Hsp70- to Hsp90-containing complexes. This leads to enhanced SRPK nuclear translocation and SR protein phosphorylation. These findings reveal a major signal transduction pathway for regulated splicing and place SRPKs in a central position in the pathway, consistent with their reputed roles in a large number of human cancers. Examination of EGF induced AKT-SRPK-SR pathway in the regulation of splicing in HEK293T cells with RASL-seq
Project description:RNA binding proteins have important functions in tumorigenesis; therefore, their regulatory mechanisms should be further explored. The present study aimed to determine the regulatory roles of RNA binding motif protein 6 (RBM6) in transcription and alternative splicing in Hela cells. RBM6 expression levels and its effect on the prognosis of different tumors in The Cancer Genome Atlas database were analyzed using TIMER2.0. Short hairpin RNA was used to ablate RBM6 expression in HeLa cells. Differentially expressed genes and alternative splicing events in Hela cells were assessed using RNA sequencing and quantitative real-time reverse transcription polymerase chain reaction. RBM6 was identified as a prognostic marker for bladder urothelial carcinoma, kidney renal clear cell carcinoma, mesothelioma, and pancreatic adenocarcinoma. Knockdown of RBM6 promoted cell proliferation. In Hela cells, RMB6 regulated the expression and alternative splicing of many genes involved in tumorigenesis_x001E_related pathways, including ‘apoptotic process’, ‘mitotic cell cycle’, and ‘RNA splicing’. RBM6-regulated alternatively spliced genes (DIABLO (Diablo IAP-binding mitochondrial protein), PAK4 (P21 (RAC1) activated kinase 4) and ERCC2 (ERCC excision repair 2, TFIIH core complex helicase subunit)) were subjected to quantitative real-time reverse transcription polymerase chain reaction validation, which demonstrated the same expression trends as the RNA sequencing results. Our findings showed that RBM6 affects tumorigenesis and cancer progression by disrupting alternative splicing in HeLa cells.
Project description:RNA-binding proteins (RBPs) have important roles in orchestrating posttranscriptional regulation and modulating many tumorigenesis events. SERBP1 has been recognized as an important regulator in multiple cancers, while it remains unclear whether SERBP1-regulated gene expression at the transcriptome-wide level is significantly correlated with tumorigenesis.We overexpressed SERBP1 in HeLa cells and explored whether SERBP1 overexpression (SERBP1-OE) affects the proliferation and apoptosis of HeLa cells. We analyzed the transcriptome-wide gene expression changes and alternative splicing changes mediated by SERBP1-OE using transcriptome sequencing method (RNA-seq). RT-qPCR was conducted to assay SERBP1-regulated alternative splicing.SERBP1-OE induced the apoptosis of HeLa cells. The downregulated genes were strongly enriched in the cell proliferation and apoptosis pathways according to the GO analysis, including FOS, FOSB, PAK6 and RAB26. The genes undergoing at least one SERBP1-regulated alternative splicing event were enriched in transcriptional regulation, suggesting a mechanism of the regulation of gene expression, and in pyruvate and fatty acid metabolic processes critical for tumorigenesis events. The SERBP1-regulated alternative splicing of ME3, LPIN3, CROT,PDP1, SLC27A1 and ALKBH7 was validated by RT-qPCR analysis.We for the first time demonstrated the cellular function and molecular targets of SERBP1 in HeLa cells at transcriptional and post-transcriptional levels. The SERBP1-regulated gene expression and alternative splicing networks revealed by this study provide important information for exploring the functional roles and regulatory mechanisms of SERBP1 in cancer development and progression.