Project description:Total RNA was extracted from C2C12 cells transfected with the indicated siRNA using TRIZOL (Thermo Fisher Scientific), and was further purified with Quick-RNA Miniprep Kit (Zymo research) according to the manufacturer’s instructions. The extracted RNA was subjected to RNA-seq at Macrogen, Japan. Briefly, a sequencing library was prepared using the TruSeq Stranded mRNA kit (Illumina), and the library was read on Illumina NovaSeq 6000 (150 bp paired-end reads).
Project description:RNA seqeuncing was performed to identifiy changes in genes expression and alternative splicing following SRSF3 depletion in pluripotent stem cells.
Project description:Ewing sarcoma is a highly aggressive tumor characterized by a translocation between members of the FET family of RNA binding proteins and one of several ETS transcription factors, with the most common translocation being EWS-FLI1. EWS-FLI1 leads to changes in gene expression through mechanisms that are not completely understood. We performed RNA sequencing analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify novel target genes. This analysis identified lnc277 as a previously uncharacterized long non-coding RNA upregulated by EWS-FLI1 in pMPCs. Inhibiting the expression of lnc277 diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar whereas inhibiting lnc277 had no effect on other cell types tested. By analyzing gene expression after shRNA knockdown, we found that both EWS-FLI1 and lnc277 repressed many more genes that they induced and that a significant fraction of EWS-FLI1 repressed targets were also repressed by lnc277. Analysis of primary human Ewing sarcoma RNA sequencing data further supports a role for lnc277 in mediating gene repression. We identified hnRNPK as an RNA binding protein that interacts directly with lnc277. We found a significant overlap in the genes repressed by hnRNPK and those repressed by both EWS-FLI1 and lnc277, suggesting that hnRNPK participates in lnc277 mediated gene repression. Thus, lnc277 is a previously uncharacterized long non-coding RNA downstream of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes. Our studies identify a novel mechanism of oncogenesis downstream of a chromosomal translocation and underscore the importance of lncRNA-mediated gene repression as a mechanism of EWS-FLI1 transcriptional regulation. A673 Ewing cells expressing an shRNA targeting hnRNPK or control were subjected to paired end RNA sequencing and compared to shGFP control.
Project description:Several members of SRSF family play wide-ranging roles in the regulation of transcription and post-splicing processes as well as splice sites selection. Although the expression of SRSF3 was reported to be overexpressed in several cancers, the roles of SRSF3 in the cancer cells are almost unknown. We analyzed differentially expressed genes in SRSF3 siRNA-treated HCT116 cells and identified the specific pathways regulated by SRSF3. After treatment of HCT116 cells with SRSF3 (sample 02) or control (sample 01) siRNA, total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA). The quality of the purified RNA and its applicability for microarray analysis were assessed by the Agilent 2100 Bioanalyzer using a RNA 6000 Nano Labchip kit (Agilent Technologies, Palo Alto, CA, USA). Total RNA (400 ng) was used for amplification, labeling and hybridization to a whole human genome oligoDNA microarray (4x44k; Agilent) according to the manufacture’s instructions.
Project description:The goal of this study is to analyse the transcriptome of control hearts vs hearts lacking SRSF3 expression and to analyse binding preferences of SRSF3 in cardiac myocytes.
Project description:Several members of SRSF family play wide-ranging roles in the regulation of transcription and post-splicing processes as well as splice sites selection. Although the expression of SRSF3 was reported to be overexpressed in several cancers, the roles of SRSF3 in the cancer cells are almost unknown. We analyzed differentially expressed genes in SRSF3 siRNA-treated HCT116 cells and identified the specific pathways regulated by SRSF3.
Project description:SRSF3 is overexpressed in human invasive ovarian cancer and its overexpression is required for cancer cell growth and survival. To decipher the mechnisms behind the role of SRSF3 in ovarian cancer, we examined the gene expression and splicing in the ovarian cancer cell line that was engineered to express a doxycycline-induced SRSF3 siRNA, which was able to knockdown SRSF3 expression by 90% and induce apoptosis. Total RNAs extracted from A2780/SRSF3si2, a subline of ovarian cancer cell line A2780, treated with or without doxycycline at 0.1ug/ml for three days were analyzed using Affymetrix GeneChip® Human Exon 1.0 ST Array